Complement Receptor 2 Targeted Complement Modulators

ABSTRACT

Modulation of the complement system represents a therapeutic modality for numerous pathologic conditions associated with complement activation. In a strategy to prepare complement inhibitors that are targeted to sites of complement activation and disease, compositions comprising a complement inhibitor linked to complement receptor (CR) 2 are disclosed. The disclosed are compositions can be used in methods of treating pathogenic diseases and inflammatory conditions by modulating the complement system.

This application claims benefit of U.S. Provisional Application No.60/426,676, filed on Nov. 15, 2002, which is incorporated herein byreference in its entirety.

I. BACKGROUND OF THE INVENTION

Complement is the collective term for a series of blood proteins and isa major effector mechanism of the immune system. Complement activationand its deposition on target structures can lead to directcomplement-mediated cell lysis, or can lead indirectly to cell or tissuedestruction due to the generation of powerful modulators of inflammationand the recruitment and activation of immune effector cells. Complementactivation products that mediate tissue injury are generated at variouspoints in the complement pathway. Inappropriate complement activation onhost tissue plays an important role in the pathology of many autoimmuneand inflammatory diseases, and is also responsible for many diseasestates associated with bioincompatibility, e.g. post-cardiopulmonaryinflammation and transplant rejection. Complement inhibition representsa potential therapeutic modality for the treatment of suchimmune-mediated diseases and disease states. Complement inhibitoryproteins that systemically inhibit complement have been shown to beeffective in various animal models of disease (and in a few clinicaltrials), but complement inhibitors that target a site of disease andcomplement activation offer significant potential advantages with regardto safety and efficacy.

In healthy individuals, complement deposition on host cell membranes isprevented by complement inhibitory proteins expressed at the cellsurface. These complement inhibitory proteins are also expressed on thesurface of tumor cells, often at increased levels, and are considered tobe an important contributing factor to the resistance of tumor cells tomonoclonal antibody-mediated immunotherapy (monoclonal antibodies thattarget to tumor cells and activate complement).

The complement system comprises a collection of about 30 proteins and isone of the major effector mechanisms of the immune system. Thecomplement cascade is activated principally via either the classical(usually antibody-dependent) or alternative (usuallyantibody-independent) pathways. Activation via either pathway leads tothe generation of C3 convertase, which is the central enzymatic complexof the cascade. C3 convertase cleaves serum C3 into C3a and C3b, thelatter of which binds covalently to the site of activation and leads tothe further generation of C3 convertase (amplification loop). Theactivation product C3b (and also C4b generated only via the classicalpathway) and its breakdown products are important opsonins and areinvolved in promoting cell-mediated lysis of target cells (by phagocytesand NK cells) as well as immune complex transport and solubilization.C3/C4 activation products and their receptors on various cells of theimmune system are also important in modulating the cellular immuneresponse. C3 convertases participate in the formation of C5 convertase,a complex that cleaves C5 to yield C5a and C5b. C5a has powerfulproinflammatory and chemotactic properties and can recruit and activateimmune effector cells. Formation of C5b initiates the terminalcomplement pathway resulting in the sequential assembly of complementproteins C6, C7, C8 and (C9)n to form the membrane attack complex (MACor C5b-9). Formation of MAC in a target cell membrane can result indirect cell lysis, but can also cause cell activation and theexpression/release of various inflammatory modulators.

There are two broad classes of membrane complement inhibitor; inhibitorsof the complement activation pathway (inhibit C3 convertase formation),and inhibitors of the terminal complement pathway (inhibit MACformation). Membrane inhibitors of complement activation includecomplement receptor 1 (CR1), decay-accelerating factor (DAF) andmembrane cofactor protein (MCP). They all have a protein structure thatconsists of varying numbers of repeating units of about 60-70 aminoacids termed short consensus repeats (SCR) that are a common feature ofC3/C4 binding proteins. Rodent homologues of human complement activationinhibitors have been identified. The rodent protein Crry is a widelydistributed inhibitor of complement activation that functions similar toboth DAF and MCP. Rodents also express DAF and MCP, although Crryappears to be functionally the most important regulator of complementactivation in rodents. Although there is no homolog of Crry found inhumans, the study of Crry and its use in animal models is clinicallyrelevant.

Control of the terminal complement pathway and MAC formation in hostcell membranes occurs principally through the activity of CD59, a widelydistributed 20 kD glycoprotein attached to plasma membranes by aglucosylphosphatidylinositol (GPI) anchor. CD59 binds to C8 and C9 inthe assembling MAC and prevents membrane insertion.

Various types of complement inhibitory proteins are currently underinvestigation for therapy of inflammatory disease and disease statesassociated with bioincompatibility. Two of the best therapeuticallycharacterized inhibitors of human complement are a soluble form ofcomplement receptor 1 (sCR1) and an anti-C5 monoclonal antibody. Thesesystemically active inhibitory proteins have shown efficacy in variousanimal models of disease and more recently in clinical trials (1-5,6:#1037). Anti-C5 mAb inhibits the generation of C5a and the MAC,whereas sCR1 is an inhibitor of complement activation and also inhibitsthe generation of C3 activation products. Soluble forms of human decayaccelerating factor (DAF) and membrane cofactor protein (MCP), membraneinhibitors of complement activation, have also been shown to beprotective an animal models of inflammation and bioincompatability(7-11). CD59 is a membrane inhibitor of complement that blocks assemblyof the MAC, but does not effect generation of complement opsonins or C3aand C5a. Soluble forms of CD59 have been produced, but its lowfunctional activity in vitro, particularly in the presence of serum,indicates that sCD59 will have little or no therapeutic efficacy(12-15).

Targeting complement inhibitors to sites of complement activation anddisease is likely to improve their efficacy. Since complement plays animportant role in host defense and immune complex catabolism, targetedcomplement inhibitors can also reduce potentially serious side effects,particularly with long term complement inhibition. Recently, a modifiedform of sCR1 decorated with sialyl Lewis x (sLex) was prepared and shownto bind to endothelial cells expressing P and E selectin. sCR1 sLex wasshown to be a more potent therapeutic than sCR1 in rodent models ofinflammatory disease (16, 17). In in vitro feasibility studies,antibody-DAF (18) and antibody-CD59 (19) fusion proteins were shown tobe more effective at protecting targeted cells than untargeted cellsfrom complement. Non-specific membrane targeting of recombinantcomplement inhibitors has also been achieved by coupling inhibitors tomembrane-inserting peptides (20, 21).

C3 activation fragments are abundant complement opsonins found at a siteof complement activation, and they serve as ligands for various C3receptors. One such receptor, complement receptor 2 (CR2), atransmembrane protein, plays an important role in humoral immunity byway of its expression predominantly on mature B cells and folliculardendritic cells (22, 23). CR2 is a member of the C3 binding proteinfamily and consists of 15-16 short consensus repeat (SCR) domains,structural units that are characteristic of these proteins, with the C3binding site being contained in the two N-terminal SCRs (24, 25). CR2 isnot an inhibitor of complement and it does not bind C3b, unlike theinhibitors of complement activation (DAF, MCP, CR1 and Crry). Naturalligands for CR2 are iC3b, C3dg and C3d, cell-bound breakdown fragmentsof C3b that bind to the two N-terminal SCR domains of CR2 (26, 27).Cleavage of C3 results initially in the generation and deposition of C3bon the activating cell surface. The C3b fragment is involved in thegeneration of enzymatic complexes that amplify the complement cascade.On a cell surface, C3b is rapidly converted to inactive iC3b,particularly when deposited on a host surface containing regulators ofcomplement activation (ie. most host tissue). Even in absence ofmembrane bound complement regulators, substantial levels of iC3b areformed. iC3b is subsequently digested to the membrane bound fragmentsC3dg and then C3d by serum proteases, but this process is relativelyslow (28, 29). Thus, the C3 ligands for CR2 are relatively long livedonce they are generated and will be present in high concentrations atsites of complement activation.

II. SUMMARY OF THE INVENTION

In accordance with the purposes of this invention, as embodied andbroadly described herein, this invention, in one aspect, relates to CR2targeted modulators of complement activity.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or can be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

III. BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and together with the description, serve to explain theprinciples of the invention.

FIG. 1 shows a diagram of examples of CR2-complement inhibitor fusionproteins.

FIG. 2 shows SDS-PAGE and Western blot analysis of purified recombinantfusion proteins and soluble complement inhibitors. Gels (10% acrylamide)were stained with coomasie blue. Western blots were developed usingantibodies to complement inhibitors as the primary antibody.

FIG. 3 shows binding of recombinant fusion proteins to C3-opsonized CHOcells. Antibody sensitized CHO cells were incubated in C6-deficientserum, washed and incubated with soluble complement inhibitor (blacktrace), or fusion protein with CR2 at N-terminus (light gray trace) orC-terminus (dark gray trace) at 20 μg/ml. Cell binding of recombinantproteins was detected by flow cytometry using anti-DAF or anti-CD59mAbs. Incubation of CHO cells with PBS instead of complement inhibitorgave similar fluorescence profile as sDAF and sCD59. Representative of 3separate experiments.

FIG. 4 shows analysis of the interaction between CR2 fusion proteins andC3d by surface plasmon resonance. Solid lines indicate differentconcentrations of CR2 fusion proteins as indicated in Figure ______[Looks like a fig. no. is missing.]. Broken lines show curves fitting toa 1:1 Langmuir binding model.

FIG. 5 shows inhibition of complement mediated lysis by recombinant sDAFand DAF fusion proteins. Antibody sensitized CHO cells (panel a) orsheep erythrocytes (panel b) were incubated with recombinant protein and10% human serum (CHO cells) or 0.33% human serum (erythrocytes). Theseconcentrations resulted in approximately 90% lysis of unprotected cells.Lysis was determined after 45 min. incubation at 37° C. Background lysisdetermined by incubating cells in heat inactivated serum was less than5% and was subtracted. Mean +/−SD, n=4.

FIG. 6 shows inhibition of complement mediated lysis by recombinantsCD59 and CD59 fusion proteins. Antibody sensitized CHO cells (panel a)or sheep erythrocytes (panel b) were incubated with recombinant proteinand 10% human serum (CHO cells) or 0.33% human serum (erythrocytes).These concentrations resulted in approximately 90% lysis of unprotectedcells. Lysis was determined after 45 min. incubation at 37° C.Background lysis determined by incubating cells in heat inactivatedserum was less than 5% and was subtracted. Mean +/−SD, n=4.

FIG. 7 shows the effect of recombinant fusion proteins on U937 celladhesion. Sheep erythrocytes were sensitized with IgM antibody andincubated in C6-deficient serum. C3 opsonized erythrocytes werecoincubated with U937 cells in the presence of 500 nM recombinant fusionprotein or PBS. Following incubation, the average number of U937 cellsbound per erythrocyte was determined my microscopy. Mean +/−SD, n=3.

FIG. 8 shows the nucleotide and predicted amino acid sequence of maturehuman CR2-DAF. Amino acids underlined represent linking sequencesbetween CR2 and DAF.

FIG. 9 shows the nucleotide and predicted amino acid sequence of maturehuman CR2-CD59. Amino acids underlined represent linking sequencesbetween CR2 and CD59.

FIG. 10 shows the nucleotide and predicted amino acid sequence of maturehuman DAF-CR2. Amino acids underlined represent linking sequencesbetween DAF and CR2.

FIG. 11 shows the nucleotide and predicted amino acid sequence of maturehuman CD59-CR2. Amino acids underlined represent linking sequencesbetween CD59 and CR2.

FIG. 12 shows targeting of CR2 containing fusion proteins to C3-coatedCHO cells. C3 ligand was generated on CHO cells by incubation of cellsin 10% anti-CHO antiserum and 10% C6-depleted human serum (to preventformation of membrane attack complex and cell lysis). Cells were washedand incubated with fusion protein (20 ug/ml, 4° C., 30 min). Binding wasdetected by flow cytometric analysis using antibodies againstappropriate complement inhibitor (DAF or CD59). Black line: control (nofusion protein); Light gray: CR2 at C-terminus; Dark gray: CR2 atN-terminus.

FIG. 13 shows analysis of CR2-DAF binding to C3dg by surface plasmonresonance.

FIG. 14 shows analysis of CR2-CD59 binding to C3dg by surface plasmonresonance.

FIG. 15 shows analysis of DAF-CR2 binding to C3dg by surface plasmonresonance.

FIG. 16 shows analysis of CD59-CR2 binding to C3dg by surface plasmonresonance.

FIG. 17 shows the effect of targeted and untargeted DAF oncomplement-mediated lysis of CHO cells. CHO cells were sensitized tocomplement with anti-CHO antisera (10% concentration, 4° C., 30 min) andsubsequently incubated with 10% normal human serum (NHS) (37° C., 60min) in the presence of varying concentrations of complement inhibitoryproteins. Cell lysis was then determined by trypan blue exclusion assay.Representative experiment showing mean +/−SD (n=3). Three separateexperiments using different fusion protein preparations performed.

FIG. 18 shows the effect of targeted and untargeted CD59 oncomplement-mediated lysis of CHO cells. Assay performed as described inlegend to FIG. 17. Representative experiment showing mean +/−SD (n=3).Three separate experiments using different fusion protein preparationsperformed.

FIG. 19 shows the effect of targeted and untargeted DAF oncomplement-mediated hemolysis. Sheep erythrocytes (E) were sensitizedwith anti-sheep E antibody and subsequently incubated with a 1/300dilution of NHS (37° C., 60 min) in the presence of varyingconcentrations of complement inhibitory proteins. Cell lysis wasdetermined by measuring released hemoglobin (absorbance at 412 nm).Representative experiment showing mean +/−SD (n=3). Two separateexperiments using different fusion protein preparations performed.

FIG. 20 shows the effect of targeted and untargeted CD59 oncomplement-mediated hemolysis. Assay performed as described in legend toFIG. 19. Representative experiment showing mean +/−SD (n=3). Twoseparate experiments using different fusion protein preparationsperformed.

FIG. 21 shows the nucleotide and predicted amino acid sequence of maturehuman CR2-human IgG1 Fc. Amino acids underlined represent linkingsequences between CR2 and Fc region. Expression plasmid contains genomicFc region (hinge-intron-CH2-intron-CH3).

FIG. 22 shows SDS-PAGE analysis of CR2-Fc fusion protein. PurifiedCR2-Fc was run under nonreducing (lane 1) or reducing (lane 2)conditions. Gel stained by coomassie blue. (for MW of markers in lane 3,see FIG. 2).

FIG. 23 shows targeting of CR2-Fc to C3-coated CHO cells. C3 ligand wasgenerated as described (legend to FIG. 12). Cells were washed andincubated with CR2-Fc (20 ug/ml, 4° C., 30 min). Binding was detected byflow cytometric analysis using antibodies against human Fc conjugated toFITC. Upper panel shows results from incubation of CR2-Fc with C3-coatedCHO cells, and lower panel shows results from incubation of CR2-Fc withcontrol CHO cells.

FIG. 24 shows surface plasmon resonance sensorgram showing binding ofCR2-Fc to C3d ligand immobilized on chip.

FIG. 25 shows the biodistribution of ¹²⁵I-CR2-DAF and ¹²⁵I-sDAF in 34week old NZB/W F1 mice. Radiolabeled proteins were injected into thetail vein and biodistribution of radiolabel determined after 24 hr. Eachprotein was injected into 2 mice.

FIG. 26 shows imaging of CR2-DAF bound to glomeruli of 24-week-oldMRL/lpr mice. Glomerular binding of CD2-DAF (a) and sDAF (b) wasanalyzed 24 hours after tail-vein injection of each protein. The figureshows immunofluorescence staining of kidney sections.

FIG. 27 shows the single chain antibody CD59-Crry construct. The figureshows the construct comprises a variable light chain (VL) and a variableheavy chain (VH) from K9/9 mAb. The construct was prepared in the yeastexpression vector pPICZalph (Invitrogen).

FIG. 28 shows the biodistribution of complement inhibitors and K9/9single chain Ab in rats. Iodinated recombinant proteins administered 4days after PAN treatment and radioactivity in organs measured 48 hrlater.

FIG. 29 shows Creatinine clearance in rats treated with PAN andreceiving indicated therapy (n=4, +/−SD).

FIG. 30 shows PAS stained renal cortex. FIG. 30A shows No PAN control,FIG. 30B: PAN with PBS treatment, FIG. 30C: PAN with targeted K9/9 Crrytreatment, and FIG. 30D: PAN with sCrry treatment.

FIG. 31 shows complement inhibitory activity in serum afteradministration of recombinant proteins. Measured by lysis of sensitizedsheep erythrocytes. Percent inhibitory activity shown relative to serumfrom control rats.

IV. DETAILED DESCRIPTION

The present invention can be understood more readily by reference to thefollowing detailed description of preferred embodiments of the inventionand the Examples included therein and to the Figures and their previousand following description.

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that thisinvention is not limited to specific synthetic methods, specificrecombinant biotechnology methods unless otherwise specified, or toparticular reagents unless otherwise specified, as such can, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only and is notintended to be limiting.

A. DEFINITIONS

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

“Treatment” or “treating” means to administer a composition to a subjectwith a condition, wherein the condition can be any pathogenic disease,autoimmune disease, cancer or inflammatory condition. The effect of theadministration of the composition to the subject can have the effect ofbut is not limited to reducing the symptoms of the condition, areduction in the severity of the condition, or the complete ablation ofthe condition.

Herein, “inhibition” or “inhibits” means to reduce activity. It isunderstood that inhibition can mean a slight reduction in activity tothe complete ablation of all activity. An “inhibitor” can be anythingthat reduces activity.

Herein, “activation” or “activates” means to increase activity. It isunderstood that activation can mean an increase in existing activity aswell as the induction of new activity. An “activator” can be anythingthat increases activity.

B. COMPLEMENT INHIBITING AND ACTIVATING CONSTRUCTS

Disclosed are compositions comprising a construct, wherein the constructcomprises CR2 and a modulator of complement activity.

CR2 consists of an extracellular portion consisting of 15 or 16repeating units known as short consensus repeats (SCRs). Amino acids1-20 comprise the leader peptide, amino acids 23-82 comprise SCR1, aminoacids 91-146 comprise SCR2, amino acids 154-210 comprise SCR3, aminoacids 215-271 comprise SCR4. The active site (C3dg binding site) islocated in SCR 1-2 (the first 2 N-terminal SCRs). SCR units areseparated by short sequences of variable length that serve as spacers.It is understood that any number of SCRs containing the active site canbe used. In one embodiment, the construct contains the 4 N-terminal SCRunits. In another embodiment, the construct includes the first twoN-terminal SCRs. In another embodiment the construct includes the firstthree N-terminal SCRs.

It is understood that species and strain variation exist for thedisclosed peptides, polypeptides, proteins, protein fragments andcompositions. Specifically disclosed are all species and strainvariations for the disclosed peptides, polypeptides, proteins, proteinfragments and compositions.

Also disclosed are compositions, wherein the construct is a fusionprotein

Herein a “fusion protein” means two or more components comprisingpeptides, polypeptides, or proteins operably linked. CR2 can be linkedto complement inhibitors or activators by an amino acid linkingsequence. Examples of linkers are well known in the art. Examples oflinkers can include but are not limited to (Gly₄Ser)₃ (G4S), (Gly₃Ser)₄(G3S), SerGly₄, and SerGly₄SerGly₄. Linking sequences can also consistof “natural” linking sequences found between SCR units within human (ormouse) proteins, for example VSVFPLE, the linking sequence between SCR 2and 3 of human CR2. Fusion proteins can also be constructed withoutlinking sequences.

Also disclosed are compositions of the invention, wherein the fusionprotein inhibits complement.

Also disclosed are compositions of the invention, wherein the modulatorof complement activity comprises a complement inhibitor.

Also disclosed are compositions of the invention; for example, whereinthe complement inhibitor is decay accelerating factor (DAF) SEQ ID NO: 1(nucleotide) and SEQ ID NO: 2 (amino acid). For example, the DAF can besoluble human DAF comprising the four SCR domains withoutglycophosphatidyl anchor and serine-threonine rich region. The DAF canalso be soluble human DAF comprising the four SCR domains and theserine-threonine rich region but without glycophosphatidyl anchor.

The DAF extracellular region consists of 4 SCR units at N-terminusfollowed by serine/threonine rich region. Amino acids 1-34 comprise theleader peptide, amino acids 35-95 comprise SCR1, amino acids 97-159comprise SCR2, amino acids 162-221 comprise SCR3, amino acids 224-284comprise SCR4, and amino acids 287-356 comprise the S/T region. In oneembodiment of the invention, the composition of the invention comprisesall 4 SCR units. In another embodiment of the invention, the compositioncomprises SCR2-4 of DAF.

Disclosed are compositions of the invention, wherein the complementinhibitor comprises a fusion protein between CD59 and another complementinhibitor selected from the group consisting of DAF, MCP, Crry and CR1.Also disclosed are compositions of the invention, wherein the complementinhibitor is a fusion protein of two or more complement inhibitors.

Also disclosed are compositions of the invention, wherein the fusionprotein comprises CR2-DAF (SEQ ID NO: 6). Also disclosed arecompositions of the invention wherein the fusion protein is encoded by anucleotide comprising SEQ ID NO: 5.

Also disclosed are compositions of the invention, wherein the fusionprotein comprises DAF-CR2 (SEQ ID NO: 10). Also disclosed arecompositions of the invention wherein the fusion protein is encoded by anucleotide comprising SEQ ID NO: 9.

Also disclosed are compositions of the invention, wherein the complementinhibitor is human CD59 (SEQ ID NO: 3 (nucleotide) and SEQ ID NO: 4(amino acid)). The human CD59 can be soluble human CD59 comprising themature protein without glycophosphatidyl anchor.

Also disclosed are compositions of the invention, wherein the fusionprotein comprises CR2-human CD59 (SEQ ID NO: 8). Also disclosed arecompositions of the invention wherein the fusion protein is encoded by anucleotide comprising SEQ ID NO: 7.

Also disclosed are compositions of the invention, wherein the fusionprotein comprises human CD59-CR2 (SEQ ID NO: 12). Also disclosed arecompositions of the invention wherein the fusion protein is encoded by anucleotide comprising SEQ ID NO: 10.

Also disclosed are compositions of the invention wherein the complementinhibitor is an antibody to C5. Also disclosed are compositions of theinvention, wherein the fusion protein comprises CR2-anti-C5 antibody.

Also disclosed are compositions of the invention, wherein the complementinhibitor is CR1 (SEQ ID NO: 13 (nucleotide) and SEQ ID NO: 14 (aminoacid)). The extracellular region of CR1 can comprise 30 SCR units. It isan embodiment of the invention that the composition can comprise theentire extracellular region of CR1. In another embodiment of theinvention, the composition comprises [the] one active site[s] of CR1.The active sites of CR1 are amino acids 1-46 which comprise the leaderpeptide, amino acids 47-300 which comprise SCR1-4 (C4b binding site,lower affinity for C3b), amino acids 497-750 which comprise SCR8-11 (C3bbinding site, lower affinity for C4b), amino acids 947-1200 whichcomprise SCR15-18 (C3b binding site, lower affinity for C4b), and aminoacids 1400-1851 which comprise the C1q binding site. In an additionalembodiment of the invention, the composition of the invention cancomprise any [one or] combination or all of the active sites of CR1.

Also disclosed are compositions of the invention, wherein the complementinhibitor comprises the active sites of CR1, and wherein [the] oneactive site[s] further comprise a leader peptide comprising amino acids6-46, amino acids 47-300 which comprise SCR1-4 (C4b binding site, loweraffinity for C3b), amino acids 497-750 which comprise SCR8-11 (C3bbinding site, lower affinity for C4b), amino acids 947-1200 whichcomprise SCR15-18 (C3b binding site, lower affinity for C4b), and aminoacids 1400-1851 which comprise the C1q binding site. In an additionalembodiment of the invention, the composition of the invention cancomprise any [one or] combination or all of the active sites of CR1.

Also disclosed are compositions of the invention, wherein the complementinhibitor is MCP (SEQ ID NO: 15 (nucleotide) and SEQ ID NO: 16 (aminoacid)). The extracellular region consists of 4 SCR units followed byser/thr region. Amino acids 1-34 comprise the leader peptide, aminoacids 35-95 comprise SCR1, amino acids 96-158 comprise SCR2, aminoacids, 159-224 comprise SCR3, amino acids 225-285 comprise SCR4, andamino acids 286-314 comprise the S/T region.

Also disclosed are compositions of the invention, wherein the complementinhibitor is Crry (SEQ ID NO: 17). The Crry can be soluble mouse Crrycomprising the 5 N-terminal SCR domains without transmembrane region.

Also disclosed are compositions of the invention, wherein the complementinhibitor is murine CD59. The murine CD59 can be soluble murine CD59comprising the mature protein without glycophosphatidyl anchor.

Disclosed are compositions of the invention, wherein the fusion proteinactivates complement.

Thus, disclosed are compositions of the invention, wherein the modulatorof complement activity comprises a complement activator.

Disclosed are compositions of the invention, wherein the complementactivator is human IgG1 Fc(SEQ ID NO: 18).

Also disclosed are compositions of the invention, wherein the complementactivator comprises CR2-human IgG1 Fc (SEQ ID NO: 20). Also disclosedare compositions of the invention wherein the fusion protein is encodedby a nucleotide comprising SEQ ID NO: 21.

Disclosed are compositions of the invention, wherein the fusion proteinis human IgM (SEQ ID NO: 19).

Also disclosed are compositions of the invention, wherein the fusionprotein comprises CR2-human IgM Fc.

Disclosed are compositions of the invention, wherein the complementactivator is mouse IgG3 (SEQ ID NO: 22).

Also disclosed are compositions of the invention, wherein the fusionprotein comprises CR2-murine IgG3 Fc.

Also disclosed are compositions of the invention, wherein the fusionprotein comprises CR2-murine IgM Fc.

It is specifically contemplated that complement activator can alsoincrease antibody-dependent cell-mediated cytotoxicity (ADCC) via the Fcportion of the composition. ADCC is the destruction of a target cell bya natural killer (NK) cell via recognition of and contact with an Fcregion and an Fc receptor on the NK cell. This can be in the form ofFcγRIII recognition of IgG1 Fc or IgG3 Fc. Following the contact of theFc receptor with the Fc, the NK cell lysis the target cell via the useof perforin and granzyme. This mechanism can be important in controllingtumor growth

Disclosed are compositions of the invention, wherein the CR2-Fc fusionprotein is not immunogenic. It is understood that a composition that isnot immunogenic (ie. does not elicit an immune response) is less likelyto be attacked and inactivated by the subjects own immune response. Theanticipated lack of CR2-Fc immunogenicity is a potential advantage overanti-C3d antibodies, even if antibodies are humanized. It is anembodiment of the invention that the Fc region fused to CR2 can be fromany human or mouse IgG isotype, human or mouse IgM, or any human ormouse IgG isotype containing a mu-tailpiece. The mu-tailpiece is an 18amino acid C-terminal region from IgM that, when added C-terminal to IgGFc sequences, results in the generation of polymeric forms of IgG(similar to IgM) that efficiently activate complement and have enhancedaffinity for Fc receptors. The fusion can occur at the hinge region ofthe Fc portion of the composition.

CR2 fusion proteins containing either IgM or IgG Fc regions with amu-tailpiece can have advantages over CR2-IgG Fc fusion proteins. IgM orIgG-mu Fc regions will result in polymeric fusion proteins with up to 6Fcs and 12 CR2 sites. These constructs can have enhanced avidity for C3ligand and enhanced effector function (complement activation and Fcreceptor binding).

Also disclosed are compositions of the invention, wherein the complementactivator is CVF (SEQ ID NO: 23 (nucleotide) and SEQ ID NO: 24 (aminoacid)).

In one embodiment of the invention, CVF can be coupled to soluble CR2.It is understood that CVF binds factor B and activates the alternativepathway of complement by forming CVFBb, a C3/C5 convertase that is notinactivated by complement inhibitory proteins. The half life of CVFBb isabout 7 hr. compared to about 1 min. for the physiological alternativepathway convertase, C3bBb.

It is an embodiment of the invention that CVF can be chemically coupledto soluble CR2.

Disclosed are compositions of the invention, wherein the construct is ina vector.

Disclosed are cells comprising the vector of the invention.

Also disclosed are compositions, wherein the construct is animmunoconjugate. Herein “immunoconjugate” means two or more componentscomprising peptides, polypeptides, or proteins operably linked by achemical cross-linker. Linking of the components of the immunoconjugatecan occur on reactive groups located on the component. Reactive groupsthat can be targeted using a cross-linker include primary amines,sulfhydryls, carbonyls, carbohydrates and carboxylic acids, or activegroups can be added to proteins. Examples of chemical linkers are wellknown in the art and can include but are not limited tobismaleimidohexane, m-maleimidobenzoyl-N-hydroxysuccinimide ester,NHS-Esters-Maleimide Crosslinkers such as MBS, Sulfo-MBS, SMPB,Sulfo-SMPB, GMBS, Sulfo-GMBS, EMCS, Sulfo-EMCS; Imidoester Cross-linkerssuch as DMA, DMP, DMS, DTBP; EDC[1-Ethyl-3-(3-Dimethylaminopropyl)carbodiimide Hydrochloride],[2-(4-Hydroxyphenyl)ethyl]-4-N-maleimidomethyl)-cyclohexane-1-carboxamide,DTME: Dithio-bis-maleimidoethane, DMA (Dimethyl adipimidate.2 HCl), DMP(Dimethyl pimelimidate.2 HCl), DMS (Dimethyl suberimidate.2 HCl), DTBP(Dimethyl 3,3′-dithiobispropionimidate.2 HCl), MBS,(m-Maleimidobenzoyl-N-hydroxysuccinimide ester), Sulfo-MBS(m-Maleimidobenzoyl-N-hydroxysuccinimide ester), Sulfo-SMPB(Sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate(, GMBS(N-[.-maleimidobutyryloxy]succinimide ester),EMCS(N-[.-maleimidocaproyloxy]succinimide ester), andSulfo-EMCS(N-[.-maleimidocaproyloxy]sulfosuccinimide ester).

C. METHODS OF USING THE COMPOSITIONS

Various types of complement inhibitory proteins are currently underinvestigation for therapy of inflammatory disease and disease statesassociated with bioincompatibility. Two of the best therapeuticallycharacterized inhibitors of human complement are a soluble form ofcomplement receptor 1 (sCR1) and an anti-C5 monoclonal antibody. Thesesystemically active inhibitory proteins have shown efficacy in variousanimal models of disease and more recently in clinical trials (1-5,6:#1037 which are incorporated herein by reference regarding teachingson in vivo efficacy and clinical results).

Disclosed are methods of treating a condition affected by complement ina subject comprising administering to the subject the composition of theinvention. It is understood that administration of the composition tothe subject can have the effect of, but is not limited to, reducing thesymptoms of the condition, a reduction in the severity of the condition,or the complete ablation of the condition.

1. Methods of Using the Compositions to Inhibit Complement

Disclosed are methods of treating a condition affected by complement ina subject comprising administering to the subject the composition of theinvention, wherein the composition will inhibit complement activity. Itis understood that the effect of the administration of the compositionto the subject can have the effect of but is not limited to reducing thesymptoms of the condition, a reduction in the severity of the condition,or the complete ablation of the condition.

Disclosed are methods of reducing complement-mediated damage comprisingadministering to a subject the composition of the invention, whichinhibits complement.

Disclosed are methods of the invention, wherein the condition treated isan inflammatory condition. Also disclosed are methods of the invention,wherein the inflammatory condition can be selected from the groupconsisting of asthma, systemic lupus erythematosus, rheumatoidarthritis, reactive arthritis, spondylarthritis, systemic vasculitis,insulin dependent diabetes mellitus, multiple sclerosis, experimentalallergic encephalomyelitis, Sjögren's syndrome, graft versus hostdisease, inflammatory bowel disease including Crohn's disease,ulcerative colitis, ischemia reperfusion injury, myocardial infarction,alzheimer's disease, transplant rejection (allogeneic and xenogeneic),thermal trauma, any immune complex-induced inflammation,glomerulonephritis, myasthenia gravis, cerebral lupus, Guillain-Barresyndrome, vasculitis, systemic sclerosis, anaphylaxis, catheterreactions, atheroma, infertility, thyroiditis, ARDS, post-bypasssyndrome, hemodialysis, juvenile rheumatoid, Behcets syndrome, hemolyticanemia, pemphigus, bullous pemphigoid, stroke, atherosclerosis, andscleroderma.

Also disclosed are methods of the invention, wherein the condition is aviral infection. Also disclosed are methods of the invention, whereinthe viral infection can be selected from the list of viruses consistingof Influenza virus A, Influenza virus B, Respiratory syncytial virus,Dengue virus, Yellow fever virus, Ebola virus, Marburg virus, Lassafever virus, Eastern Equine Encephalitis virus, Japanese Encephalitisvirus, St. Louis Encephalitis virus, Murray Valley fever virus, WestNile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, RotavirusC, Sindbis virus, Hantavirus.

Disclosed are methods of the invention, wherein the condition is aninflammatory respose to a viral vector. The viral vector can be selectedfrom the list of viruses consisting of adenovirus, vaccinia virus, adenoassociated virus, modified vaccinia ancara virus, and cytomegliavirus.It is understood that other viral vectors are in use for vaccinedelivery. Specifically disclosed are each and every viral vector knownin the art.

It is understood in the art that Candida express a CR3 like protein thathas similar binding properties as CR2. The CR3 like protein appears tobe involved in pathogenesis. Therefore, an embodiment of the inventionis treating a subject with a fungal infection, wherein the treatmentblocks fungal-“CR3” function as well as inhibits complement, comprisingadministering to a subject the composition of the invention.

Disclosed are methods of the invention, wherein complement inhibitor canenhance the outcome of apoptosis-base therapy (e.g., gene therapy withadenovirus expressing Fas ligand).

Apoptosis occurring during normal development is non inflammatory and isinvolved in induction of immunological tolerance. Although apoptoticcell death can be inflammatory depending on how it is activated and inwhat cell types (for example, therapeutic agents that ligate Fas areable to induce inflammation), necrotic cell death results in a sustainedand powerful inflammatory response mediated by released cell contentsand by proinflammatory cytokines released by stimulated phagocytes.Apoptotic cells and vesicles are normally cleared by phagocytes, thuspreventing the pro-inflammatory consequences of cell lysis. In thiscontext, it has been shown that apoptotic cells and apoptotic bodiesdirectly fix complement, and that complement can sustain ananti-inflammatory response due to opsonization and enhanced phagocytosisof apoptotic cells.

Inflammation is involved in non specific recruitment of immune cellsthat can influence innate and adaptive immune responses. Modulatingcomplement activation during apoptosis-based tumor therapy to inhibitphagocytic uptake of apoptotic cells/bodies enhances theinflammatory/innate immune response within the tumor environment. Inaddition, apoptotic cells can be a source of immunogenic self antigensand uncleared apoptotic bodies can result in autoimmunization. Inaddition to creating an enhanced immuno-stimulatory environment,modulating complement at a site in which tumor cells have been inducedto undergo apoptosis further augments or triggers specific immunityagainst a tumor to which the host is normally tolerant.

The disclosed compositions of the invention can act as CR2 and CR3antagonists. Disclosed are methods of inhibiting complement activity viainhibition of CR2 comprising administering the composition of theinvention to a subject. Also disclosed are methods of inhibitingcomplement activity via inhibition of CR3 comprising administering thecomposition of the invention to a subject. As a CR2 antagonist canmodulate immune response, a CR3 antagonist can have secondanti-inflammatory mechanism of action since CR3 is integrin that bindsendothelial ICAM1. ICAM1 is expressed at sites of inflammation and isinvolved in leukocyte adhesion and diapedesis. In addition, ICAM1expression is upregulated by complement activation products.

2. Methods of Using the Compositions to Activate Complement

Disclosed are methods of treating a condition affected by complement ina subject comprising administering to the subject the composition of theinvention, wherein the composition will activate complement. It isunderstood that the administration of the composition to the subject canhave the effect of, but is not limited to, reducing the symptoms of thecondition, a reduction in the severity of the condition, or the completeablation of the condition.

Disclosed are methods of enhancing complement-mediated damage comprisingadministering to a subject the composition of the invention, whichactivates complement.

Also disclosed are methods of the invention, wherein the condition is acancer. The cancer can be selected from the group consisting oflymphomas (Hodgkins and non-Hodgkins), B cell lymphoma, T cell lymphoma,myeloid leukemia, leukemias, mycosis fungoides, carcinomas, carcinomasof solid tissues, squamous cell carcinomas, adenocarcinomas, sarcomas,gliomas, blastomas, neuroblastomas, plasmacytomas, histiocytomas,melanomas, adenomas, hypoxic tumours, myelomas, AIDS-related lymphomasor sarcomas, metastatic cancers, bladder cancer, brain cancer, nervoussystem cancer, squamous cell carcinoma of head and neck,neuroblastoma/glioblastoma, ovarian cancer, skin cancer, liver cancer,melanoma, squamous cell carcinomas of the mouth, throat, larynx, andlung, colon cancer, cervical cancer, cervical carcinoma, breast cancer,epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer,esophageal carcinoma, head and neck carcinoma, hematopoietic cancers,testicular cancer, colo-rectal cancers, prostatic cancer, or pancreaticcancer.

In one embodiment of the invention CR2 can target complement depositedon tumor cells as a result of administered anti-tumor antibodies, or asa result of a normally ineffective humoral immune response.

Thus the present complement activating composition can be administeredin conjunction with anti-tumor antibodies. Examples of such anti-tumorantibodies are well known and include anti-PSMA monoclonal antibodiesJ591, PEQ226.5, and PM2P079.1 (Fracasso, G. et al., (2002) Prostate53(1): 9-23); anti-Her2 antibody hu4D5 (Gerstner, R. B., et al., (2002)J. Mol. Biol. 321(5): 851-62); anti-disialosyl Gb5 monoclonal antibody5F3 which can be used as an anti renal cell carcinoma antibody (Ito A.et al., (2001) Glycoconj. J. 18(6): 475-485); anti MAGE monoclonalantibody 57B (Antonescu, C. R. et al., (2002) Hum. Pathol. 33(2):225-9); anti-cancer monoclonal antibody CLN-Ig (Kubo, O. et al., (2002)Nippon Rinsho. 60(3): 497-503); anti-Dalton's lymphoma associatedantigen (DLAA) monoclonal antibody DLAB (Subbiah, K. et al., (2001)Indian J. Exp. Biol. 39(10): 993-7). The present composition can beadministered before, concurrent with or after administration of theanti-tumor antibody, so long as the present composition is present atthe tumor during the time when the antibody is also present at thetumor.

A representative but non-limiting list of cancers that the disclosedcompositions can be used to treat is the following: lymphoma, B celllymphoma, T cell lymphoma, mycosis fungoides, multiple myeloma,Hodgkin's Disease, myeloid leukemia, bladder cancer, brain cancer,nervous system cancer, head and neck cancer, squamous cell carcinoma ofhead and neck, kidney cancer, lung cancers such as small cell lungcancer and non-small cell lung cancer, urothelial carcinomas,adenocarcinomas, sarcomas, gliomas, high grade gliomas, blastomas,neuroblastomas, plasmacytomas, histiocytomas, adenomas, hypoxic tumours,myelomas, AIDS-related lymphomas or sarcomas, metastatic cancers,neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer, prostatecancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas ofthe mouth, throat, larynx, and lung, colon cancer, cervical cancer,cervical carcinoma, breast cancer, and epithelial cancer, renal cancer,genitourinary cancer, pulmonary cancer, esophageal carcinoma, head andneck carcinoma, large bowel cancer, hematopoietic cancers; testicularcancer; colon and rectal cancers, stomach cancer, prostatic cancer,Waldenstroms disease or pancreatic cancer.

The complement activating compositions disclosed herein can also be usedfor the treatment of precancer conditions such as cervical and analdysplasias, other dysplasias, severe dysplasias, hyperplasias, atypicalhyperplasias, and neoplasias. Disclosed are methods of the invention,wherein the condition is a precancer conditions. It is understood thatthe composition will recognize antigens that are overexpressed on thesurface of precancerous cells

Also disclosed are methods of using the complement activatingcompositions of the invention to treat viral infection. The viralinfection can be selected from the list of viruses consisting of Herpessimplex virus type-1, Herpes simplex virus type-2, Cytomegalovirus,Epstein-Barr virus, Varicella-zoster virus, Human herpesvirus 6, Humanherpesvirus 7, Human herpesvirus 8, Variola virus, Vesicular stomatitisvirus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus,Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus, Influenzavirus A, Influenza virus B, Measles virus, Polyomavirus, HumanPapilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus,Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus,Yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus,Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St.Louis Encephalitis virus, Murray Valley fever virus, West Nile virus,Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbisvirus, Simian Immunodeficiency virus, Human T-cell Leukemia virustype-1, Hantavirus, Rubella virus, Simian Immunodeficiency virus, HumanImmunodeficiency virus type-1, and Human Immunodeficiency virus type-2.

Also disclosed are methods of using the complement activatingcompositions of the invention to treat a bacterial infection. Alsodisclosed are methods of the invention, wherein the bacterial infectioncan be selected from the list of bacterium consisting of M.tuberculosis, M bovis, M. bovis strain BCG, BCG substrains, M. avium, M.intracellulare, M. africanum, M. kansasii, M. marinum, M ulcerans, M.avium subspecies paratuberculosis, Nocardia asteroides, other Nocardiaspecies, Legionella pneumophila, other Legionella species, Salmonellatyphi, other Salmonella species, Shigella species, Yersinia pestis,Pasteurella haemolytica, Pasteurella multocida, other Pasteurellaspecies, Actinobacillus pleuropneumoniae, Listeria monocytogenes,Listeria ivanovii, Brucella abortus, other Brucella species, Cowdriaruminantium, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydiapsittaci, Coxiella burnetti, other Rickettsial species, Ehrlichiaspecies, Staphylococcus aureus, Staphylococcus epidermidis,Streptococcus pyogenes, Streptococcus agalactiae, Bacillus anthracis,Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserriameningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, otherPseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, otherHemophilus species, Clostridium tetani, other Clostridium species,Yersinia enterolitica, and other Yersinia species.

Also disclosed are methods of using the complement activatingcompositions of the invention to treat a parasitic infection. Alsodisclosed are methods of the invention, wherein the parasitic infectioncan be selected from the group consisting of Toxoplasma gondii,Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, otherPlasmodium species., Trypanosoma brucei, Trypanosoma cruzi, Leishmaniamajor, other Leishmania species., Schistosoma mansoni, other Schistosomaspecies., and Entamoeba histolytica.

Also disclosed are methods of using the complement activatingcompositions of the invention to treat a fungal infection. Alsodisclosed are methods of the invention, wherein the fungal infection canbe selected from the group consisting of Candida albicans, Cryptococcusneoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodesimmitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis,Pneomocystis carnii, Penicillium marneffi, and Alternaria alternata. Inthe methods of the invention, the subject can be a mammal. For example,the mammal can be a human, nonhuman primate, mouse, rat, pig, dog, cat,monkey, cow, or horse.

3. Methods of Using the Compositions as Research Tools

The disclosed compositions can be used in a variety of ways as researchtools. For example, the disclosed compositions can be used to studyinhibitor of complement activation.

The disclosed compositions can be used as diagnostic tools related todiseases associated with complement activation, such as cancer, viralinfections, bacterial infections, parasitic infections, and fungalinfections. CR2-fusion proteins will target a site of complementactivation and a labeled CR2-fusion protein can diagnose conditionsassociated with complement activation. For example, a tumor-reactiveantibody would activate complement on tumor cells, which CR2 could thentarget. The labeled CR2-Fc could then amplify the signal followingantibody targeting.

D. COMPOSITIONS

Disclosed are the components to be used to prepare the disclosedcompositions as well as the compositions themselves to be used in themethods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference for each of the various individual and collectivecombinations and permutation of these compounds may not be explicitlymade, each is specifically contemplated and described herein. Forexample, if a particular CR2, DAF, CD59, CR1, MCP, Crry, IgG1, IgM,IgG3, CVF is described, and/or a specific combination thereof isdisclosed and discussed and/or a number of modifications that can bemade to a number of molecules including the CR2, DAF, CD59, CR1, MCP,Crry, IgG1, IgM, IgG3, CVF, and/or combination thereof are discussed,specifically contemplated is each and every combination and permutationof CR2, DAF, CD59, CR1, MCP, Crry, IgG1, IgM, IgG3, CVF, or combinationthereof and the modifications that are possible, unless specificallyindicated to the contrary. Thus, if a class of molecules A, B, and C aredisclosed as well as a class of molecules D, E, and F and an example ofa combination molecule, A-D is disclosed, then even if each is notindividually recited each is individually and collectively contemplatedmeaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F areconsidered disclosed. Likewise, any subset or combination of these isalso disclosed. Thus, for example, the sub-group of A-E, B-F, and C-Ewould be considered disclosed. This concept applies to all aspects ofthis application including, but not limited to, steps in methods ofmaking and using the disclosed compositions. Thus, if there are avariety of additional steps that can be performed it is understood thateach of these additional steps can be performed with any specificembodiment or combination of embodiments of the disclosed methods.

1. Sequence Similarities

It is understood that as discussed herein the use of the terms homologyand identity mean the same thing as similarity. Thus, for example, ifthe use of the word homology is used between two non-natural sequencesit is understood that this is not necessarily indicating an evolutionaryrelationship between these two sequences, but rather is looking at thesimilarity or relatedness between their nucleic acid sequences. Many ofthe methods for determining homology between two evolutionarily relatedmolecules are routinely applied to any two or more nucleic acids orproteins for the purpose of measuring sequence similarity regardless ofwhether they are evolutionarily related or not.

In general, it is understood that one way to define any known variantsand derivatives or those that might arise, of the genes and proteinsdisclosed herein, is through defining the variants and derivatives interms of homology to specific known sequences. This identity ofparticular sequences disclosed herein is also discussed elsewhereherein. For example SEQ ID NO: 25 sets forth a particular sequence of aCR2 and SEQ ID NO: 26 sets forth a particular sequence of the proteinencoded by SEQ ID NO: 25, a CR2 protein. Specifically disclosed arevariants of these and other genes and proteins herein disclosed whichhave at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percenthomology to the stated sequence. Those of skill in the art readilyunderstand how to determine the homology of two proteins or nucleicacids, such as genes. For example, the homology can be calculated afteraligning the two sequences so that the homology is at its highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison can beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment. It isunderstood that any of the methods typically can be used and that incertain instances the results of these various methods can differ, butthe skilled artisan understands if identity is found with at least oneof these methods, the sequences would be said to have the statedidentity, and be disclosed herein.

For example, as used herein, a sequence recited as having a particularpercent homology to another sequence refers to sequences that have therecited homology as calculated by any one or more of the calculationmethods described above. For example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingthe Zuker calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by any of theother calculation methods. As another example, a first sequence has 80percent homology, as defined herein, to a second sequence if the firstsequence is calculated to have 80 percent homology to the secondsequence using both the Zuker calculation method and the Pearson andLipman calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by the Smith andWaterman calculation method, the Needleman and Wunsch calculationmethod, the Jaeger calculation methods, or any of the other calculationmethods. As yet another example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingeach of calculation methods (although, in practice, the differentcalculation methods will often result in different calculated homologypercentages).

2. Nucleic Acids

There are a variety of molecules disclosed herein that are nucleic acidbased, including for example the nucleic acids that encode, for exampleCR2, DAF, CD59, CR1, MCP, Crry, IgG1, IgM, IgG3, CVF, CR2-DAF, DAF-CR2,CR2-CD59, CD59-CR2, CR2-CR1, CR1-CR2, CR2-MCP, MCP-CR2, CR2-Crry,Crry-CR2, CR2-anti-C5, CR2-IgG1 Fc (human), CR2-IgM Fc, CR2-IgG3 Fc(murine), or CR2-CVF, as well as various functional nucleic acids. Thedisclosed nucleic acids are made up of for example, nucleotides,nucleotide analogs, or nucleotide substitutes. Non-limiting examples ofthese and other molecules are discussed herein. It is understood thatfor example, when a vector is expressed in a cell, that the expressedmRNA will typically be made up of A, C, G, and U. Likewise, it isunderstood that if, for example, an antisense molecule is introducedinto a cell or cell environment through for example exogenous delivery,it is advantageous that the antisense molecule be made up of nucleotideanalogs that reduce the degradation of the antisense molecule in thecellular environment.

a) Nucleotides and Related Molecules

A nucleotide is a molecule that contains a base moiety, a sugar moietyand a phosphate moiety. Nucleotides can be linked together through theirphosphate moieties and sugar moieties creating an internucleosidelinkage. The base moiety of a nucleotide can be adenin-9-yl (A),cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T).The sugar moiety of a nucleotide is a ribose or a deoxyribose. Thephosphate moiety of a nucleotide is pentavalent phosphate. Annon-limiting example of a nucleotide would be 3′-AMP (3′-adenosinemonophosphate) or 5′-GMP (5′-guanosine monophosphate).

A nucleotide analog is a nucleotide which contains some type ofmodification to either the base, sugar, or phosphate moieties.Modifications to the base moiety would include natural and syntheticmodifications of A, C, G, and T/U as well as different purine orpyrimidine bases, such as uracil-5-yl (.psi.), hypoxanthin-9-yl (I), and2-aminoadenin-9-yl. A modified base includes but is not limited to5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Additional basemodifications can be found for example in U.S. Pat. No. 3,687,808,Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and Sanghvi, Y. S., Chapter 15, Antisense Research andApplications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRCPress, 1993. Certain nucleotide analogs, such as 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine can increase the stability of duplex formation. Oftentime base modifications can be combined with for example a sugarmodification, such as 2′-O-methoxyethyl, to achieve unique propertiessuch as increased duplex stability. There are numerous United Statespatents such as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, which detail and describe a range of basemodifications. Each of these patents is herein incorporated byreference.

Nucleotide analogs can also include modifications of the sugar moiety.Modifications to the sugar moiety would include natural modifications ofthe ribose and deoxy ribose as well as synthetic modifications. Sugarmodifications include but are not limited to the following modificationsat the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—,S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl andalkynyl can be substituted or unsubstituted C₁ to C₁₀, alkyl or C₂ toC₁₀ alkenyl and alkynyl. 2′ sugar modifications also include but are notlimited to —O[(CH₂)_(n)O]_(m)CH₃, —O(CH₂)_(n)OCH₃, —O(CH₂)_(n)NH₂,—O(CH₂)_(n)CH₃, —O(CH₂)_(n)—ONH₂, and —O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂,where n and m are from 1 to about 10.

Other modifications at the 2′ position include but are not limited to:C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. Similar modifications canalso be made at other positions on the sugar, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide. Modifiedsugars would also include those that contain modifications at thebridging ring oxygen, such as CH₂ and S. Nucleotide sugar analogs canalso have sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. There are numerous United States patents thatteach the preparation of such modified sugar structures such as U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, each of which is hereinincorporated by reference in its entirety.

Nucleotide analogs can also be modified at the phosphate moiety.Modified phosphate moieties include but are not limited to those thatcan be modified so that the linkage between two nucleotides contains aphosphorothioate, chiral phosphorothioate, phosphorodithioate,phosphotriester, aminoalkylphosphotriester, methyl and other alkylphosphonates including 3′-alkylene phosphonate and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates. It is understood that these phosphate or modifiedphosphate linkage between two nucleotides can be through a 3′-5′ linkageor a 2′-5′ linkage, and the linkage can contain inverted polarity suchas 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and freeacid forms are also included. Numerous United States patents teach howto make and use nucleotides containing modified phosphates and includebut are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is hereinincorporated by reference.

It is understood that nucleotide analogs need only contain a singlemodification, but can also contain multiple modifications within one ofthe moieties or between different moieties.

Nucleotide substitutes are molecules having similar functionalproperties to nucleotides, but which do not contain a phosphate moiety,such as peptide nucleic acid (PNA). Nucleotide substitutes are moleculesthat will recognize nucleic acids in a Watson-Crick or Hoogsteen manner,but which are linked together through a moiety other than a phosphatemoiety. Nucleotide substitutes are able to conform to a double helixtype structure when interacting with the appropriate target nucleicacid.

Nucleotide substitutes are nucleotides or nucleotide analogs that havehad the phosphate moiety and/or sugar moieties replaced. Nucleotidesubstitutes do not contain a standard phosphorus atom. Substitutes forthe phosphate can be for example, short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatom and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts. Numerous United States patents disclosehow to make and use these types of phosphate replacements and includebut are not limited to U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439,each of which is herein incorporated by reference.

It is also understood in a nucleotide substitute that both the sugar andthe phosphate moieties of the nucleotide can be replaced, by for examplean amide type linkage (aminoethylglycine) (PNA). U.S. Pat. Nos.5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNAmolecules, each of which is herein incorporated by reference. (See alsoNielsen et al., Science, 1991, 254, 1497-1500).

It is also possible to link other types of molecules (conjugates) tonucleotides or nucleotide analogs to enhance for example, cellularuptake. Conjugates can be chemically linked to the nucleotide ornucleotide analogs. Such conjugates include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al.,Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al.,FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937. Numerous United States patents teach thepreparation of such conjugates and include, but are not limited to U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,each of which is herein incorporated by reference.

A Watson-Crick interaction is at least one interaction with theWatson-Crick face of a nucleotide, nucleotide analog, or nucleotidesubstitute. The Watson-Crick face of a nucleotide, nucleotide analog, ornucleotide substitute includes the C2, N1, and C6 positions of a purinebased nucleotide, nucleotide analog, or nucleotide substitute and theC2, N3, C4 positions of a pyrimidine based nucleotide, nucleotideanalog, or nucleotide substitute.

A Hoogsteen interaction is the interaction that takes place on theHoogsteen face of a nucleotide or nucleotide analog, which is exposed inthe major groove of duplex DNA. The Hoogsteen face includes the N7position and reactive groups (NH2 or O) at the C6 position of purinenucleotides.

b) Sequences

There are a variety of sequences related to the CR2, DAF, CD59, CR1,MCP, Crry, IgG1, IgM, IgG3, CVF, CR2-DAF, DAF-CR2, CR2-CD59, CD59-CR2,CR2-CR1, CR1-CR2, CR2-MCP, MCP-CR2, CR2-Crry, Crry-CR2, CR2-IgG1 Fc(human), CR2-IgM Fc, CR2-IgG3 Fc (murine), or CR2-CVF genes having, forexample, the sequences as disclosed herein or sequences available in theliterature. These sequences and others are herein incorporated byreference in their entireties as well as for individual subsequencescontained therein.

One particular sequence set forth in SEQ ID NO: 25 used herein, as anexample, to exemplify the disclosed compositions and methods. It isunderstood that the description related to this sequence is applicableto any sequence related to CR2, CR2-DAF, DAF-CR2, CR2-CD59, CD59-CR2,CR2-CR1, CR1-CR2, CR2-MCP, MCP-CR2, CR2-Crry, Crry-CR2, CR2-IgG1 Fc(human), CR2-IgM Fc, CR2-IgG3 Fc (murine), or CR2-CVF unlessspecifically indicated otherwise. Those of skill in the art understandhow to resolve sequence discrepancies and differences and to adjust thecompositions and methods relating to a particular sequence to otherrelated sequences (i.e. sequences of CR2, DAF, CD59, CR1, MCP, Crry,IgG1, IgM, IgG3, CVF, CR2-DAF, DAF-CR2, CR2-CD59, CD59-CR2, CR2-CR1,CR1-CR2, CR2-MCP, MCP-CR2, CR2-Crry, Crry-CR2, CR2-IgG1 Fc (human),CR2-IgM Fc, CR2-IgG3 Fc (murine), or CR2-CVF). Primers and/or probes canbe designed for any CR2, DAF, CD59, CR1, MCP, Crry, IgG1, IgM, IgG3,CVF, CR2-DAF, DAF-CR2, CR2-CD59, CD59-CR2, CR2-CR1, CR1-CR2, CR2-MCP,MCP-CR2, CR2-Crry, Crry-CR2, CR2-IgG1 Fc (human), CR2-IgM Fc, CR2-IgG3Fc (murine), or CR2-CVF sequence given the information disclosed hereinand known in the art.

3. Delivery of the Compositions to Cells

There are a number of compositions and methods which can be used todeliver the present fusion protein compositions, immunoconjugatecompositions, and nucleic acid compositions to cells, either in vitro orin vivo. Compositions of the invention are preferably administered to asubject in a pharmaceutically acceptable carrier. Suitable carriers andtheir formulations are described in Remington: The Science and Practiceof Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company,Easton, Pa. 1995. Typically, an appropriate amount of apharmaceutically-acceptable salt is used in the formulation to renderthe formulation isotonic. Examples of the pharmaceutically-acceptablecarriers include, but are not limited to, saline, water:oil emulsions,oil:water emulsions, water:oil:water emulsions, and Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The compositions of the invention can be administered to the subject,patient, or cell by injection (e.g., intravenous, intraperitoneal,subcutaneous, intramuscular), or by other methods such as infusion thatensure its delivery to the bloodstream in an effective form. Local orintravenous injection is preferred.

Effective dosages and schedules for administering the compositions ofthe invention can be determined empirically, and making suchdeterminations is within the skill in the art. Those skilled in the artwill understand that the dosage of the compositions of the inventionthat must be administered will vary depending on, for example, thesubject that will receive the composition, the route of administration,the particular type of composition used and other drugs beingadministered. A typical daily dosage of the compositions of theinvention used alone might range from about 1 μg/kg to up to 100 mg/kgof body weight or more per day, depending on the factors mentionedabove.

a) Nucleic Acid Based Delivery Systems

There are a number of compositions and methods which can be used todeliver nucleic acids to cells, either in vitro or in vivo. Thesemethods and compositions can largely be broken down into two classes:viral based delivery systems and non-viral based delivery systems. Forexample, the nucleic acids can be delivered through a number of directdelivery systems such as, electroporation, lipofection, calciumphosphate precipitation, plasmids, viral vectors, viral nucleic acids,phage nucleic acids, phages, cosmids, or via transfer of geneticmaterial in cells or carriers such as cationic liposomes. Appropriatemeans for transfection, including viral vectors, chemical transfectants,or physico-mechanical methods such as electroporation and directdiffusion of DNA, are described by, for example, Wolff, J. A., et al.,Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818,(1991) Such methods are well known in the art and readily adaptable foruse with the compositions and methods described herein. In certaincases, the methods will be modified to specifically function with largeDNA molecules. Further, these methods can be used to target certaindiseases and cell populations by using the targeting characteristics ofthe carrier.

Transfer vectors can be any nucleotide construction used to delivergenes into cells (e.g., a plasmid), or as part of a general strategy todeliver genes, e.g., as part of recombinant retrovirus or adenovirus(Ram et al. Cancer Res. 53:83-88, (1993)).

As used herein, plasmid or viral vectors are agents that transport thedisclosed nucleic acids, such as SEQ ID NO: 25 into the cell withoutdegradation and include a promoter yielding expression of the gene inthe cells into which it is delivered. In some embodiments the CR2, DAF,CD59, CR1, MCP, Crry, IgG1, IgM, IgG3, CVF, CR2-DAF, DAF-CR2, CR2-CD59,CD59-CR2, CR2-CR1, CR1-CR2, CR2-MCP, MCP-CR2, CR2-Crry, Crry-CR2,CR2-IgG1 Fc (human), CR2-IgM Fc, CR2-IgG3 Fc (murine), or CR2-CVFs arederived from either a virus or a retrovirus. Viral vectors are, forexample, Adenovirus, Adeno-associated virus, Herpes virus, Vacciniavirus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis andother RNA viruses, including these viruses with the HIV backbone. Alsopreferred are any viral families which share the properties of theseviruses which make them suitable for use as vectors. Retrovirusesinclude Murine Maloney Leukemia virus, MMLV, and retroviruses thatexpress the desirable properties of M V as a vector. Retroviral vectorsare able to carry a larger genetic payload, i.e., a transgene or markergene, than other viral vectors, and for this reason are a commonly usedvector. However, they are not as useful in non-proliferating cells.Adenovirus vectors are relatively stable and easy to work with, havehigh titers, and can be delivered in aerosol formulation, and cantransfect non-dividing cells. Pox viral vectors are large and haveseveral sites for inserting genes, they are thermostable and can bestored at room temperature. A preferred embodiment is a viral vectorwhich has been engineered so as to suppress the immune response of thehost organism, elicited by the viral antigens. Preferred vectors of thistype will carry coding regions for Interleukin 8 or 10.

Viral vectors can have higher transaction (ability to introduce genes)abilities than chemical or physical methods to introduce genes intocells. Typically, viral vectors contain, nonstructural early genes,structural late genes, an RNA polymerase III transcript, invertedterminal repeats necessary for replication and encapsidation, andpromoters to control the transcription and replication of the viralgenome. When engineered as vectors, viruses typically have one or moreof the early genes removed and a gene or gene/promotor cassette isinserted into the viral genome in place of the removed viral DNA.Constructs of this type can carry up to about 8 kb of foreign geneticmaterial. The necessary functions of the removed early genes aretypically supplied by cell lines which have been engineered to expressthe gene products of the early genes in trans.

(1) Retroviral Vectors

A retrovirus is an animal virus belonging to the virus family ofRetroviridae, including any types, subfamilies, genus, or tropisms.Retroviral vectors, in general, are described by Verma, I. M.,Retroviral vectors for gene transfer. In Microbiology-1985, AmericanSociety for Microbiology, pp. 229-232, Washington, (1985), which isincorporated by reference herein. Examples of methods for usingretroviral vectors for gene therapy are described in U.S. Pat. Nos.4,868,116 and 4,980,286; PCT applications WO 90/02806 and WO 89/07136;and Mulligan, (Science 260:926-932 (1993)); the teachings of which areincorporated herein by reference.

A retrovirus is essentially a package which has packed into it nucleicacid cargo. The nucleic acid cargo carries with it a packaging signal,which ensures that the replicated daughter molecules will be efficientlypackaged within the package coat. In addition to the package signal,there are a number of molecules which are needed in cis, for thereplication, and packaging of the replicated virus. Typically aretroviral genome, contains the gag, pol, and env genes which areinvolved in the making of the protein coat. It is the gag, pol, and envgenes which are typically replaced by the foreign DNA that it is to betransferred to the target cell. Retrovirus vectors typically contain apackaging signal for incorporation into the package coat, a sequencewhich signals the start of the gag transcription unit, elementsnecessary for reverse transcription, including a primer binding site tobind the tRNA primer of reverse transcription, terminal repeat sequencesthat guide the switch of RNA strands during DNA synthesis, a purine richsequence 5′ to the 3′ LTR that serve as the priming site for thesynthesis of the second strand of DNA synthesis, and specific sequencesnear the ends of the LTRs that enable the insertion of the DNA state ofthe retrovirus to insert into the host genome. The removal of the gag,pol, and env genes allows for about 8 kb of foreign sequence to beinserted into the viral genome, become reverse transcribed, and uponreplication be packaged into a new retroviral particle. This amount ofnucleic acid is sufficient for the delivery of a one to many genesdepending on the size of each transcript. It is preferable to includeeither positive or negative selectable markers along with other genes inthe insert.

Since the replication machinery and packaging proteins in mostretroviral vectors have been removed (gag, pol, and env), the vectorsare typically generated by placing them into a packaging cell line. Apackaging cell line is a cell line which has been transfected ortransformed with a retrovirus that contains the replication andpackaging machinery, but lacks any packaging signal. When the vectorcarrying the DNA of choice is transfected into these cell lines, thevector containing the gene of interest is replicated and packaged intonew retroviral particles, by the machinery provided in cis by the helpercell. The genomes for the machinery are not packaged because they lackthe necessary signals.

(2) Adenoviral Vectors

The construction of replication-defective adenoviruses has beendescribed (Berkner et al., J. Virology 61:1213-1220 (1987); Massie etal., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et al., J. Virology57:267-274 (1986); Davidson et al., J. Virology 61:1226-1239 (1987);Zhang “Generation and identification of recombinant adenovirus byliposome-mediated transfection and PCR analysis” BioTechniques15:868-872 (1993)). The benefit of the use of these viruses as vectorsis that they are limited in the extent to which they can spread to othercell types, since they can replicate within an initial infected cell,but are unable to form new infectious viral particles. Recombinantadenoviruses have been shown to achieve high efficiency gene transferafter direct, in vivo delivery to airway epithelium, hepatocytes,vascular endothelium, CNS parenchyma and a number of other tissue sites(Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin.Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092(1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle, Science259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992);Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout,Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993);Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen.Virology 74:501-507 (1993)). Recombinant adenoviruses achieve genetransduction by binding to specific cell surface receptors, after whichthe virus is internalized by receptor-mediated endocytosis, in the samemanner as wild type or replication-defective adenovirus (Chardonnet andDales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985);Seth, et al., J. Virol. 51:650-655 (1984); Seth, et al., Mol. Cell.Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991);Wickham et al., Cell 73:309-319 (1993)).

A viral vector can be one based on an adenovirus which has had the E1gene removed and these virons are generated in a cell line such as thehuman 293 cell line. In another preferred embodiment both the E1 and E3genes are removed from the adenovirus genome.

(3) Adeno-Asscociated Viral Vectors

Another type of viral vector is based on an adeno-associated virus(AAV). This defective parvovirus is a preferred vector because it caninfect many cell types and is nonpathogenic to humans. AAV type vectorscan transport about 4 to 5 kb and wild type AAV is known to stablyinsert into chromosome 19. Vectors which contain this site specificintegration property are preferred. An especially preferred embodimentof this type of vector is the P4.1 C vector produced by Avigen, SanFrancisco, Calif., which can contain the herpes simplex virus thymidinekinase gene, HSV-tk, and/or a marker gene, such as the gene encoding thegreen fluorescent protein, GFP.

In another type of AAV virus, the AAV contains a pair of invertedterminal repeats (ITRs) which flank at least one cassette containing apromoter which directs cell-specific expression operably linked to aheterologous gene. Heterologous in this context refers to any nucleotidesequence or gene which is not native to the AAV or B19 parvovirus.

Typically the AAV and B19 coding regions have been deleted, resulting ina safe, noncytotoxic vector. The AAV ITRs, or modifications thereof,confer infectivity and site-specific integration, but not cytotoxicity,and the promoter directs cell-specific expression. U.S. Pat. No.6,261,834 is herein incorporated by reference for material related tothe AAV vector.

The vectors of the present invention thus provide DNA molecules whichare capable of integration into a mammalian chromosome withoutsubstantial toxicity.

The inserted genes in viral and retroviral usually contain promoters,and/or enhancers to help control the expression of the desired geneproduct. A promoter is generally a sequence or sequences of DNA thatfunction when in a relatively fixed location in regard to thetranscription start site. A promoter contains core elements required forbasic interaction of RNA polymerase and transcription factors, and cancontain upstream elements and response elements.

(4) Large Payload Viral Vectors

Molecular genetic experiments with large human herpesviruses haveprovided a means whereby large heterologous DNA fragments can be cloned,propagated and established in cells permissive for infection withherpesviruses (Sun et al., Nature genetics 8: 33-41, 1994; Cotter andRobertson, Curr Opin Mol Ther 5: 633-644, 1999). These large DNA viruses(herpes simplex virus (HSV) and Epstein-Barr virus (EBV), have thepotential to deliver fragments of human heterologous DNA>150 kb tospecific cells. EBV recombinants can maintain large pieces of DNA in theinfected B-cells as episomal DNA. Individual clones carried humangenomic inserts up to 330 kb appeared genetically stable The maintenanceof these episomes requires a specific EBV nuclear protein, EBNA1,constitutively expressed during infection with EBV. Additionally, thesevectors can be used for transfection, where large amounts of protein canbe generated transiently in vitro. Herpesvirus amplicon systems are alsobeing used to package pieces of DNA>220 kb and to infect cells that canstably maintain DNA as episomes.

Other useful systems include, for example, replicating andhost-restricted non-replicating vaccinia virus vectors.

b) Non-Nucleic Acid Based Systems

The disclosed compositions can be delivered to the target cells in avariety of ways. For example, the compositions can be delivered throughelectroporation, or through lipofection, or through calcium phosphateprecipitation. The delivery mechanism chosen will depend in part on thetype of cell targeted and whether the delivery is occurring for examplein vivo or in vitro.

Thus, the compositions can comprise, in addition to the disclosed CR2,DAF, CD59, CR1, MCP, Crry, IgG1, IgM, IgG3, CVF, CR2-DAF, DAF-CR2,CR2-CD59, CD59-CR2, CR2-CR1, CR1-CR2, CR2-MCP, MCP-CR2, CR2-Crry,Crry-CR2, CR2-IgG1 Fc (human), CR2-IgM Fc, CR2-IgG3 Fc (murine), orCR2-CVF or vectors for example, lipids such as liposomes, such ascationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionicliposomes. Liposomes can further comprise proteins to facilitatetargeting a particular cell, if desired. Administration of a compositioncomprising a compound and a cationic liposome can be administered to theblood afferent to a target organ or inhaled into the respiratory tractto target cells of the respiratory tract. Regarding liposomes, see,e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989);Felgner et al. Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987); U.S. Pat.No. 4,897,355. Furthermore, the compound can be administered as acomponent of a microcapsule that can be targeted to specific cell types,such as macrophages, or where the diffusion of the compound or deliveryof the compound from the microcapsule is designed for a specific rate ordosage.

In the methods described above which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), delivery of the compositions to cells canbe via a variety of mechanisms. As one example, delivery can be via aliposome, using commercially available liposome preparations such asLIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.),SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (PromegaBiotec, Inc., Madison, Wis.), as well as other liposomes developedaccording to procedures standard in the art. In addition, the nucleicacid or vector of this invention can be delivered in vivo byelectroporation, the technology for which is available from Genetronics,Inc. (San Diego, Calif.) as well as by means of a SONOPORATION machine(ImaRx Pharmaceutical Corp., Tucson, Ariz.).

The materials can be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These can be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter, et al., BioconjugateChem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281,(1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, etal., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., CancerImmunol. Immunother. 35:421-425, (1992); Pietersz and McKenzie,Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). These techniques can be used for avariety of other specific cell types. Vehicles such as “stealth” andother antibody conjugated liposomes (including lipid mediated drugtargeting to colonic carcinoma), receptor mediated targeting of DNAthrough cell specific ligands, lymphocyte directed tumor targeting, andhighly specific therapeutic retroviral targeting of murine glioma cellsin vivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

Nucleic acids that are delivered to cells which are to be integratedinto the host cell genome, typically contain integration sequences.These sequences are often viral related sequences, particularly whenviral based systems are used. These viral intergration systems can alsobe incorporated into nucleic acids which are to be delivered using anon-nucleic acid based system of deliver, such as a liposome, so thatthe nucleic acid contained in the delivery system can be come integratedinto the host genome.

Other general techniques for integration into the host genome include,for example, systems designed to promote homologous recombination withthe host genome. These systems typically rely on sequence flanking thenucleic acid to be expressed that has enough homology with a targetsequence within the host cell genome that recombination between thevector nucleic acid and the target nucleic acid takes place, causing thedelivered nucleic acid to be integrated into the host genome. Thesesystems and the methods necessary to promote homologous recombinationare known to those of skill in the art.

c) In Vivo/Ex Vivo

As described above, the compositions can be administered in apharmaceutically acceptable carrier and can be delivered to thesubject=s cells in vivo and/or ex vivo by a variety of mechanisms wellknown in the art (e.g., uptake of naked DNA, liposome fusion,intramuscular injection of DNA via a gene gun, endocytosis and thelike).

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The compositions can be introduced into the cells via anygene transfer mechanism, such as, for example, calcium phosphatemediated gene delivery, electroporation, microinjection orproteoliposomes. The transduced cells can then be infused (e.g., in apharmaceutically acceptable carrier) or homotopically transplanted backinto the subject per standard methods for the cell or tissue type.Standard methods are known for transplantation or infusion of variouscells into a subject.

4. Expression Systems

The nucleic acids that are delivered to cells typically containexpression controlling systems. For example, the inserted genes in viraland retroviral systems usually contain promoters, and/or enhancers tohelp control the expression of the desired gene product. A promoter isgenerally a sequence or sequences of DNA that function when in arelatively fixed location in regard to the transcription start site. Apromoter contains core elements required for basic interaction of RNApolymerase and transcription factors, and can contain upstream elementsand response elements.

a) Viral Promoters And Enhancers

Preferred promoters controlling transcription from vectors in mammalianhost cells can be obtained from various sources, for example, thegenomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis-B virus and most preferably cytomegalovirus, orfrom heterologous mammalian promoters, e.g. beta actin promoter. Theearly and late promoters of the SV40 virus are conveniently obtained asan SV40 restriction fragment which also contains the SV40 viral originof replication (Fiers et al., Nature, 273: 113 (1978)). The immediateearly promoter of the human cytomegalovirus is conveniently obtained asa HindIII E restriction fragment (Greenway, P. J. et al., Gene 18:355-360 (1982)). Of course, promoters from the host cell or relatedspecies also are useful herein.

Enhancer generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′(Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3′(Lusky, M. L., et al., Mol. Cell. Bio. 3: 1108 (1983)) to thetranscription unit. Furthermore, enhancers can be within an intron(Banerji, J. L. et al., Cell 33: 729 (1983)) as well as within thecoding sequence itself (Osborne, T. F., et al., Mol. Cell. Bio. 4: 1293(1984)). They are usually between 10 and 300 bp in length, and theyfunction in cis. Enhancers function to increase transcription fromnearby promoters. Enhancers also often contain response elements thatmediate the regulation of transcription. Promoters can also containresponse elements that mediate the regulation of transcription.Enhancers often determine the regulation of expression of a gene. Whilemany enhancer sequences are now known from mammalian genes (globin,elastase, albumin, -fetoprotein and insulin), typically one will use anenhancer from a eukaryotic cell virus for general expression. Preferredexamples are the SV40 enhancer on the late side of the replicationorigin (bp 100-270), the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

The promotor and/or enhancer can be specifically activated either bylight or specific chemical events which trigger their function. Systemscan be regulated by reagents such as tetracycline and dexamethasone.There are also ways to enhance viral vector gene expression by exposureto irradiation, such as gamma irradiation, or alkylating chemotherapydrugs.

In certain embodiments the promoter and/or enhancer region can act as aconstitutive promoter and/or enhancer to maximize expression of theregion of the transcription unit to be transcribed. In certainconstructs the promoter and/or enhancer region be active in alleukaryotic cell types, even if it is only expressed in a particular typeof cell at a particular time. A preferred promoter of this type is theCMV promoter (650 bases). Other preferred promoters are SV40 promoters,cytomegalovirus (full length promoter), and retroviral vector LTF.

It has been shown that all specific regulatory elements can be clonedand used to construct expression vectors that are selectively expressedin specific cell types such as melanoma cells. The glial fibrillaryacetic protein (GFAP) promoter has been used to selectively expressgenes in cells of glial origin.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells) can also contain sequencesnecessary for the termination of transcription which can affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′ untranslated regions also include transcription termination sites. Itis preferred that the transcription unit also contains a polyadenylationregion. One benefit of this region is that it increases the likelihoodthat the transcribed unit will be processed and transported like mRNA.The identification and use of polyadenylation signals in expressionconstructs is well established. It is preferred that homologouspolyadenylation signals be used in the transgene constructs. In certaintranscription units, the polyadenylation region is derived from the SV40early polyadenylation signal and consists of about 400 bases. It is alsopreferred that the transcribed units contain other standard sequencesalone or in combination with the above sequences improve expressionfrom, or stability of, the construct.

b) Markers

The viral vectors can include nucleic acid sequence encoding a markerproduct. This marker product is used to determine if the gene has beendelivered to the cell and once delivered is being expressed. Preferredmarker genes are the E. Coli lacZ gene, which encodes β-galactosidase,and green fluorescent protein.

In some embodiments the marker can be a selectable marker. Examples ofsuitable selectable markers for mammalian cells are dihydrofolatereductase (DHFR), thymidine kinase, neomycin, neomycin analog G418,hydromycin, and puromycin. When such selectable markers are successfullytransferred into a mammalian host cell, the transformed mammalian hostcell can survive if placed under selective pressure. There are twowidely used distinct categories of selective regimes. The first categoryis based on a cell's metabolism and the use of a mutant cell line whichlacks the ability to grow independent of a supplemented media. Twoexamples are: CHO DHFR-cells and mouse LTK-cells. These cells lack theability to grow without the addition of such nutrients as thymidine orhypoxanthine. Because these cells lack certain genes necessary for acomplete nucleotide synthesis pathway, they cannot survive unless themissing nucleotides are provided in a supplemented media. An alternativeto supplementing the media is to introduce an intact DHFR or TK geneinto cells lacking the respective genes, thus altering their growthrequirements. Individual cells which were not transformed with the DHFRor TK gene will not be capable of survival in non-supplemented media.

The second category is dominant selection which refers to a selectionscheme used in any cell type and does not require the use of a mutantcell line. These schemes typically use a drug to arrest growth of a hostcell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, (Southern P. and Berg,P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan,R. C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B.et al., Mol. Cell. Biol. 5: 410-413 (1985)). The three examples employbacterial genes under eukaryotic control to convey resistance to theappropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid)or hygromycin, respectively. Others include the neomycin analog G418 andpuramycin.

5. Peptides

a) Protein Variants

As discussed herein there are numerous variants of the CR2, DAF, CD59,CR1, MCP, Crry, IgG1, IgM, IgG3, CVF, CR2-DAF, DAF-CR2, CR2-CD59,CD59-CR2, CR2-CR1, CR1-CR2, CR2-MCP, MCP-CR2, CR2-Crry, Crry-CR2,CR2-IgG1 Fc (human), CR2-IgM Fc, CR2-IgG3 Fc (murine), and CR2-CVFprotein that are known and herein contemplated. In addition, to theknown functional CR2, DAF, CD59, CR1, MCP, Crry, IgG1, IgM, IgG3, CVF,CR2-DAF, DAF-CR2, CR2-CD59, CD59-CR2, CR2-CR1, CR1-CR2, CR2-MCP,MCP-CR2, CR2-Crry, Crry-CR2, CR2-IgG1 Fc (human), CR2-IgM Fc, CR2-IgG3Fc (murine), and CR2-CVF strain variants, there are derivatives of theCR2, DAF, CD59, CR1, MCP, Crry, IgG1, IgM, IgG3, CVF, CR2-DAF, DAF-CR2,CR2-CD59, CD59-CR2, CR2-CR1, CR1-CR2, CR2-MCP, MCP-CR2, CR2-Crry,Crry-CR2, CR2-IgG1 Fc (human), CR2-IgM Fc, CR2-IgG3 Fc (murine), andCR2-CVF proteins which also function in the disclosed methods andcompositions. Protein variants and derivatives are well understood tothose of skill in the art and can involve amino acid sequencemodifications. For example, amino acid sequence modifications typicallyfall into one or more of three classes: substitutional, insertional ordeletional variants. Insertions include amino and/or carboxyl terminalfusions as well as intrasequence insertions of single or multiple aminoacid residues. Insertions ordinarily will be smaller insertions thanthose of amino or carboxyl terminal fusions, for example, on the orderof one to four residues. Immunogenic fusion protein derivatives, such asthose described in the examples, are made by fusing a polypeptidesufficiently large to confer immunogenicity to the target sequence bycross-linking in vitro or by recombinant cell culture transformed withDNA encoding the fusion. Deletions are characterized by the removal ofone or more amino acid residues from the protein sequence. Typically, nomore than about from 2 to 6 residues are deleted at any one site withinthe protein molecule. These variants ordinarily are prepared by sitespecific mutagenesis of nucleotides in the DNA encoding the protein,thereby producing DNA encoding the variant, and thereafter expressingthe DNA in recombinant cell culture. Techniques for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown, for example M13 primer mutagenesis and PCR mutagenesis. Aminoacid substitutions are typically of single residues, but can occur at anumber of different locations at once; insertions usually will be on theorder of about from 1 to 10 amino acid residues; and deletions willrange about from 1 to 30 residues. Deletions or insertions preferablyare made in adjacent pairs, i.e. a deletion of 2 residues or insertionof 2 residues. Substitutions, deletions, insertions or any combinationthereof can be combined to arrive at a final construct. The mutationsmust not place the sequence out of reading frame and preferably will notcreate complementary regions that could produce secondary mRNAstructure. Substitutional variants are those in which at least oneresidue has been removed and a different residue inserted in its place.Such substitutions generally are made in accordance with the followingTables 1 and 2 and are referred to as conservative substitutions.

TABLE 1 Amino Acid Abbreviations Amino Acid Abbreviations alanine Ala Aallosoleucine AIle arginine Arg R asparagine Asn N aspartic acid Asp Dcysteine Cys C glutamic acid Glu E glutamine Gln Q glycine Gly Ghistidine His H isolelucine Ile I leucine Leu L lysine Lys Kphenylalanine Phe F proline Pro P pyroglutamic pGlu acidp serine Ser Sthreonine Thr T tyrosine Tyr Y tryptophan Trp W valine Val V

TABLE 2 Amino Acid Substitutions Original Residue Exemplary ConservativeSubstitutions, others are known in the art. Ala; Ser Arg; Lys; Gln Asn;Gln; His Asp; Glu Cys; Ser Gln; Asn, Lys Glu; Asp Gly; Pro His; Asn; GlnIle; Leu; Val Leu; Ile; Val Lys; Arg; Gln; Met; Leu; Ile Phe; Met; Leu;Tyr Ser; Thr Thr; Ser Trp; Tyr Tyr; Trp; Phe Val; Ile; Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in Table2, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site or (c) the bulk of the side chain. The substitutions whichin general are expected to produce the greatest changes in the proteinproperties will be those in which (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine, in this case, (e) by increasing the number of sites forsulfation and/or glycosylation.

For example, the replacement of one amino acid residue with another thatis biologically and/or chemically similar is known to those skilled inthe art as a conservative substitution. For example, a conservativesubstitution would be replacing one hydrophobic residue for another, orone polar residue for another. The substitutions include combinationssuch as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser,Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variationsof each explicitly disclosed sequence are included within the mosaicpolypeptides provided herein.

Substitutional or deletional mutagenesis can be employed to insert sitesfor N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).Deletions of cysteine or other labile residues also may be desirable.Deletions or substitutions of potential proteolysis sites, e.g. Arg, isaccomplished for example by deleting one of the basic residues orsubstituting one by glutaminyl or histidyl residues.

Certain post-translational derivatizations are the result of the actionof recombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and asparyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Otherpost-translational modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the o-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco pp 79-86[1983]), acetylation of the N-terminal amine and, in some instances,amidation of the C-terminal carboxyl.

It is understood that one way to define the variants and derivatives ofthe disclosed proteins herein is through defining the variants andderivatives in terms of homology/identity to specific known sequences.For example, SEQ ID NO: 26 sets forth a particular sequence of CR2 andSEQ ID NO: 2 sets forth a particular sequence of a DAF protein.Specifically disclosed are variants of these and other proteins hereindisclosed which have at least, 70% or 75% or 80% or 85% or 90% or 95%homology to the stated sequence. Those of skill in the art readilyunderstand how to determine the homology of two proteins. For example,the homology can be calculated after aligning the two sequences so thatthe homology is at its highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison can beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. MoL. Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment.

It is understood that the description of conservative mutations andhomology can be combined together in any combination, such asembodiments that have at least 70% homology to a particular sequencewherein the variants are conservative mutations.

As this specification discusses various proteins and protein sequencesit is understood that the nucleic acids that can encode those proteinsequences are also disclosed. This would include all degeneratesequences related to a specific protein sequence, i.e. all nucleic acidshaving a sequence that encodes one particular protein sequence as wellas all nucleic acids, including degenerate nucleic acids, encoding thedisclosed variants and derivatives of the protein sequences. Thus, whileeach particular nucleic acid sequence may not be written out herein, itis understood that each and every sequence is in fact disclosed anddescribed herein through the disclosed protein sequence. For example,one of the many nucleic acid sequences that can encode the proteinsequence set forth in SEQ ID NO:26 is set forth in SEQ ID NO:25. Inaddition, for example, a disclosed conservative derivative of SEQ IDNO:26 is shown in SEQ ID NO: 29, where the isoleucine (I) at position 9is changed to a valine (V). It is understood that for this mutation allof the nucleic acid sequences that encode this particular derivative ofany of the disclosed sequences are also disclosed. It is also understoodthat while no amino acid sequence indicates what particular DNA sequenceencodes that protein within an organism, where particular variants of adisclosed protein are disclosed herein, the known nucleic acid sequencethat encodes that protein from which that protein arises is also knownand herein disclosed and described.

6. Antibodies

a) Antibodies Generally

The term “antibodies” is used herein in a broad sense and includes bothpolyclonal and monoclonal antibodies. In addition to intactimmunoglobulin molecules, also included in the term “antibodies” arefragments or polymers of those immunoglobulin molecules, and human orhumanized versions of immunoglobulin molecules or fragments thereof, asdescribed herein. The antibodies are tested for their desired activityusing the in vitro assays described herein, or by analogous methods,after which their in vivo therapeutic and/or prophylactic activities aretested according to known clinical testing methods.

As used herein, the term “antibody” encompasses, but is not limited to,whole immunoglobulin (i.e., an intact antibody) of any class. Nativeantibodies are usually heterotetrameric glycoproteins, composed of twoidentical light (L) chains and two identical heavy (H) chains.Typically, each light chain is linked to a heavy chain by one covalentdisulfide bond, while the number of disulfide linkages varies betweenthe heavy chains of different immunoglobulin isotypes. Each heavy andlight chain also has regularly spaced intrachain disulfide bridges. Eachheavy chain has at one end a variable domain (V(H)) followed by a numberof constant domains. Each light chain has a variable domain at one end(V(L)) and a constant domain at its other end; the constant domain ofthe light chain is aligned with the first constant domain of the heavychain, and the light chain variable domain is aligned with the variabledomain of the heavy chain. Particular amino acid residues are believedto form an interface between the light and heavy chain variable domains.The light chains of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa (k) andlambda (l), based on the amino acid sequences of their constant domains.Depending on the amino acid, sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of human immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Oneskilled in the art would recognize the comparable classes for mouse. Theheavy chain constant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively.

The term “variable” is used herein to describe certain portions of thevariable domains that differ in sequence among antibodies and are usedin the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not usually evenlydistributed through the variable domains of antibodies. It is typicallyconcentrated in three segments called complementarity determiningregions (CDRs) or hypervariable regions both in the light chain and theheavy chain variable domains. The more highly conserved portions of thevariable domains are called the framework (FR). The variable domains ofnative heavy and light chains each comprise four FR regions, largelyadopting a b-sheet configuration, connected by three CDRs, which formloops connecting, and in some cases forming part of, the b-sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the antigen binding site of antibodies (see Kabat E. A.et al., “Sequences of Proteins of Immunological Interest,” NationalInstitutes of Health, Bethesda, Md. (1987)). The constant domains arenot involved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

As used herein, the term “antibody or fragments thereof” encompasseschimeric antibodies and hybrid antibodies, with dual or multiple antigenor epitope specificities, and fragments, such as scFv, sFv, F(ab′)2,Fab′, Fab and the like, including hybrid fragments. Thus, fragments ofthe antibodies that retain the ability to bind their specific antigensare provided. For example, fragments of antibodies which maintaincomplement binding activity binding activity are included within themeaning of the term “antibody or fragment thereof.” Such antibodies andfragments can be made by techniques known in the art and can be screenedfor specificity and activity according to the methods set forth in theExamples and in general methods for producing antibodies and screeningantibodies for specificity and activity (See Harlow and Lane.Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, NewYork, (1988)).

Also included within the meaning of “antibody or fragments thereof” areconjugates of antibody fragments and antigen binding proteins (singlechain antibodies) as described, for example, in U.S. Pat. No. 4,704,692,the contents of which are hereby incorporated by reference.

As used herein, the term “antibody” or “antibodies” can also refer to ahuman antibody and/or a humanized antibody. Many non-human antibodies(e.g., those derived from mice, rats, or rabbits) are naturallyantigenic in humans, and thus can give rise to undesirable immuneresponses when administered to humans. Therefore, the use of human orhumanized antibodies in the methods of the invention serves to lessenthe chance that an antibody administered to a human will evoke anundesirable immune response.

Optionally, the antibodies are generated in other species and“humanized” for administration in humans. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fc, scFv, sFv, Fv, Fab, Fab′,F(ab′)2, or other antigen-binding subsequences of antibodies) whichcontain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies can also comprise residues thatare found neither in the recipient antibody nor in the imported CDR orframework sequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-327 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important in order to reduceantigenicity. According to the “best-fit” method, the sequence of thevariable domain of a rodent antibody is screened against the entirelibrary of known human variable domain sequences. The human sequencewhich is closest to that of the rodent is then accepted as the humanframework (FR) for the humanized antibody (Sims et al., J. Immunol.,151:2296 (1993) and Chothia et al., J. Mol. Biol., 196:901 (1987)).Another method uses a particular framework derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework can be used for several differenthumanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products using threedimensional models of the parental and humanized sequences. Threedimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequence so that thedesired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding(see, WO 94/04679, published 3 Mar. 1994).

The Fab fragments produced in the antibody digestion also contain theconstant domains of the light chain and the first constant domain of theheavy chain. Fab′ fragments differ from Fab fragments by the addition ofa few residues at the carboxy terminus of the heavy chain domainincluding one or more cysteines from the antibody hinge region. TheF(ab′)2 fragment is a bivalent fragment comprising two Fab′ fragmentslinked by a disulfide bridge at the hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. Antibody fragments originallywere produced as pairs of Fab′ fragments which have hinge cysteinesbetween them. Other chemical couplings of antibody fragments are alsoknown.

An isolated immunogenically specific paratope or fragment of theantibody is also provided. A specific immunogenic epitope of theantibody can be isolated from the whole antibody by chemical ormechanical disruption of the molecule. The purified fragments thusobtained are tested to determine their immunogenicity and specificity bythe methods taught herein. Immunoreactive paratopes of the antibody,optionally, are synthesized directly. An immunoreactive fragment isdefined as an amino acid sequence of at least about two to fiveconsecutive amino acids derived from the antibody amino acid sequence.

One method of producing proteins comprising the antibodies of thepresent invention is to link two or more peptides or polypeptidestogether by protein chemistry techniques. For example, peptides orpolypeptides can be chemically synthesized using currently availablelaboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) orBoc (tert-butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc.,Foster City, Calif.). One skilled in the art can readily appreciate thata peptide or polypeptide corresponding to the antibody of the presentinvention, for example, can be synthesized by standard chemicalreactions. For example, a peptide or polypeptide can be synthesized andnot cleaved, from its synthesis resin whereas the other fragment of anantibody can be synthesized and subsequently cleaved from the resin,thereby exposing a terminal group which is functionally blocked on theother fragment. By peptide condensation reactions, these two fragmentscan be covalently joined via a peptide bond at their carboxyl and aminotermini, respectively, to form an antibody, or fragment thereof. (GrantG A (1992) Synthetic Peptides: A User Guide. W.H. Freeman and Co., N.Y.(1992); Bodansky M and Trost B., Ed. (1993) Principles of PeptideSynthesis. Springer-Verlag Inc., NY. Alternatively, the peptide orpolypeptide is independently synthesized in vivo as described above.Once isolated, these independent peptides or polypeptides can be linkedto form an antibody or fragment thereof via similar peptide condensationreactions.

For example, enzymatic ligation of cloned or synthetic peptide segmentsallow relatively short peptide fragments to be joined to produce largerpeptide fragments, polypeptides or whole protein domains (Abrahmsen L etal., Biochemistry, 30:4151 (1991)). Alternatively, native chemicalligation of synthetic peptides can be utilized to syntheticallyconstruct large peptides or polypeptides from shorter peptide fragments.This method consists of a two step chemical reaction (Dawson et al.Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779(1994)). The first step is the chemoselective reaction of an unprotectedsynthetic peptide-alpha-thioester with another unprotected peptidesegment containing an amino-terminal Cys residue to give athioester-linked intermediate as the initial covalent product. Without achange in the reaction conditions, this intermediate undergoesspontaneous, rapid intramolecular reaction to form a native peptide bondat the ligation site. Application of this native chemical ligationmethod to the total synthesis of a protein molecule is illustrated bythe preparation of human interleukin 8 (IL-8) (Baggiolini M et al.(1992) FEBS Lett. 307:97-101; Clark-Lewis I et al., J. Biol. Chem.,269:16075 (1994); Clark-Lewis I et al., Biochemistry, 30:3128 (1991);Rajarathnam K et al., Biochemistry 33:6623-30 (1994)).

Alternatively, unprotected peptide segments are chemically linked wherethe bond formed between the peptide segments as a result of the chemicalligation is an unnatural (non-peptide) bond (Schnolzer, M et al.Science, 256:221 (1992)). This technique has been used to synthesizeanalogs of protein domains as well as large amounts of relatively pureproteins with full biological activity (deLisle Milton R C et al.,Techniques in Protein Chemistry IV. Academic Press, New York, pp.257-267 (1992)).

The invention also provides fragments of antibodies which havebioactivity. The polypeptide fragments of the present invention can berecombinant proteins obtained by cloning nucleic acids encoding thepolypeptide in an expression system capable of producing the polypeptidefragments thereof, such as an adenovirus or baculovirus expressionsystem. For example, one can determine the active domain of an antibodyfrom a specific hybridoma that can cause a biological effect associatedwith the interaction of the antibody with an Fc receptor. For example,amino acids found to not contribute to either the activity or thebinding specificity or affinity of the antibody can be deleted without aloss in the respective activity. For example, in various embodiments,amino or carboxy-terminal amino acids are sequentially removed fromeither the native or the modified non-immunoglobulin molecule or theimmunoglobulin molecule and the respective activity assayed in one ofmany available assays. In another example, a fragment of an antibodycomprises a modified antibody wherein at least one amino acid has beensubstituted for the naturally occurring amino acid at a specificposition, and a portion of either amino terminal or carboxy terminalamino acids, or even an internal region of the antibody, has beenreplaced with a polypeptide fragment or other moiety, such as biotin,which can facilitate in the purification of the modified antibody.

The fragments, whether attached to other sequences or not, includeinsertions, deletions, substitutions, or other selected modifications ofparticular regions or specific amino acids residues, provided theactivity of the fragment is not significantly altered or impairedcompared to the nonmodified antibody or antibody fragment. Thesemodifications can provide for some additional property, such as toremove or add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody can be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antigen. (Zoller M J et al.Nucl. Acids Res. 10:6487-500 (1982).

A variety of immunoassay formats can be used to select antibodies thatselectively bind with a particular protein, variant, or fragment. Forexample, solid-phase ELISA immunoassays are routinely used to selectantibodies selectively immunoreactive with a protein, protein variant,or fragment thereof. See Harlow and Lane. Antibodies, A LaboratoryManual. Cold Spring Harbor Publications, New York, (1988), for adescription of immunoassay formats and conditions that could be used todetermine selective binding. The binding affinity of a monoclonalantibody can, for example, be determined by the Scatchard analysis ofMunson et al., Anal. Biochem., 107:220 (1980).

Also provided is an antibody reagent kit comprising containers of themonoclonal antibody or fragment thereof of the invention and one or morereagents for detecting binding of the antibody or fragment thereof tothe Fc receptor molecule. The reagents can include, for example,fluorescent tags, enzymatic tags, or other tags. The reagents can alsoinclude secondary or tertiary antibodies or reagents for enzymaticreactions, wherein the enzymatic reactions produce a product that can bevisualized.

b) Human Antibodies

The human antibodies of the invention can be prepared using anytechnique. Examples of techniques for human monoclonal antibodyproduction include those described by Cole et al. (Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77, 1985) and by Boerner et al. (J.Immunol., 147(1):86-95, 1991). Human antibodies of the invention (andfragments thereof) can also be produced using phage display libraries(Hoogenboom et al., J. Mol. Biol., 227:381, 1991; Marks et al., J. Mol.Biol., 222:581, 1991).

The human antibodies of the invention can also be obtained fromtransgenic animals. For example, transgenic, mutant mice that arecapable of producing a full repertoire of human antibodies, in responseto immunization, have been described (see, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al.,Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33(1993)). Specifically, the homozygous deletion of the antibody heavychain joining region (J(H)) gene in these chimeric and germ-line mutantmice results in complete inhibition of endogenous antibody production,and the successful transfer of the human germ-line antibody gene arrayinto such germ-line mutant mice results in the production of humanantibodies upon antigen challenge. Antibodies having the desiredactivity are selected using Env-CD4-co-receptor complexes as describedherein.

c) Administration of Antibodies

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Suitable carriers and theirformulations are described in Remington: The Science and Practice ofPharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company, Easton,Pa. 1995. Typically, an appropriate amount of apharmaceutically-acceptable salt is used in the formulation to renderthe formulation isotonic. Examples of the pharmaceutically-acceptablecarrier include, but are not limited to, saline, Ringer's solution anddextrose solution. The pH of the solution is preferably from about 5 toabout 8, and more preferably from about 7 to about 7.5. Further carriersinclude sustained release preparations such as semipermeable matrices ofsolid hydrophobic polymers containing the antibody, which matrices arein the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies can bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. Guidance in selecting appropriate doses forantibodies is found in the literature on therapeutic uses of antibodies,e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., NogesPublications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith etal., Antibodies in Human Diagnosis and Therapy, Haber et al., eds.,Raven Press, New York (1977) pp. 365-389. A typical daily dosage of theantibody used alone might range from about 1 μg/kg to up to 100 mg/kg ofbody weight or more per day, depending on the factors mentioned above.

Following administration of an antibody for treating, inhibiting, orpreventing an HIV infection, the efficacy of the therapeutic antibodycan be assessed in various ways well known to the skilled practitioner.For instance, one of ordinary skill in the art will understand that anantibody of the invention is efficacious in treating or inhibiting anHIV infection in a subject by observing that the antibody reduces viralload or prevents a further increase in viral load. Viral loads can bemeasured by methods that are known in the art, for example, usingpolymerase chain reaction assays to detect the presence of HIV nucleicacid or antibody assays to detect the presence of HIV protein in asample (e.g., but not limited to, blood) from a subject or patient, orby measuring the level of circulating anti-HIV antibody levels in thepatient. Efficacy of the antibody treatment can also be determined bymeasuring the number of CD4⁺T cells in the HIV-infected subject. Anantibody treatment that inhibits an initial or further decrease in CD4⁺Tcells in an HIV-positive subject or patient, or that results in anincrease in the number of CD4⁺T cells in the HIV-positive subject, is anefficacious antibody treatment.

d) Nucleic Acid Approaches for Antibody Delivery

The compositions of the invention can also be administered to patientsor subjects as a nucleic acid preparation (e.g., DNA or RNA) thatencodes the antibody or antibody fragment, such that the patient's orsubject's own cells take up the nucleic acid and produce and secrete theencoded composition (e.g., CR2-DAF, DAF-CR2, CR2-CD59, CD59-CR2,CR2-CR1, CR1-CR2, CR2-MCP, MCP-CR2, CR2-Crry, Crry-CR2, CR2-IgG1 Fc(human), CR2-IgM Fc, CR2-IgG3 Fc (murine), or CR2-CVF).

e) Nucleic Acid Delivery

In the methods described above which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), the nucleic acids of the presentinvention can be in the form of naked DNA or RNA, or the nucleic acidscan be in a vector for delivering the nucleic acids to the cells,whereby the antibody-encoding DNA fragment is under the transcriptionalregulation of a promoter, as would be well understood by one of ordinaryskill in the art. The vector can be a commercially availablepreparation, such as an adenovirus vector (Quantum Biotechnologies, Inc.(Laval, Quebec, Canada). Delivery of the nucleic acid or vector to cellscan be via a variety of mechanisms. As one example, delivery can be viaa liposome, using commercially available liposome preparations such asLIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.),SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (PromegaBiotec, Inc., Madison, Wis.), as well as other liposomes developedaccording to procedures standard in the art. In addition, the nucleicacid or vector of this invention can be delivered in vivo byelectroporation, the technology for which is available from Genetronics,Inc. (San Diego, Calif.) as well as by means of a SONOPORATION machine(ImaRx Pharmaceutical Corp., Tucson, Ariz.).

As one example, vector delivery can be via a viral system, such as aretroviral vector system which can package a recombinant retroviralgenome (see e.g., Pastan et al., Proc. Natl. Acad. Sci. U.S.A. 85:4486,1988; Miller et al., Mol. Cell. Biol. 6:2895, 1986). The recombinantretrovirus can then be used to infect and thereby deliver to theinfected cells nucleic acid encoding a broadly neutralizing antibody (oractive fragment thereof) of the invention. The exact method ofintroducing the altered nucleic acid into mammalian cells is, of course,not limited to the use of retroviral vectors. Other techniques arewidely available for this procedure including the use of adenoviralvectors (Mitani et al., Hum. Gene Ther. 5:941-948, 1994),adeno-associated viral (AAV) vectors (Goodman et al., Blood84:1492-1500, 1994), lentiviral vectors (Naidini et al., Science272:263-267, 1996), pseudotyped retroviral vectors (Agrawal et al.,Exper. Hematol. 24:738-747, 1996). Physical transduction techniques canalso be used, such as liposome delivery and receptor-mediated and otherendocytosis mechanisms (see, for example, Schwartzenberger et al., Blood87:472-478, 1996). This invention can be used in conjunction with any ofthese or other commonly used gene transfer methods.

As one example, if the complement modulating construct-encoding nucleicacid of the invention is delivered to the cells of a subject in anadenovirus vector, the dosage for administration of adenovirus to humanscan range from about 10⁷ to 10⁹ plaque forming units (pfu) per injectionbut can be as high as 10¹² pfu per injection (Crystal, Hum. Gene Ther.8:985-1001, 1997; Alvarez and Curiel, Hum. Gene Ther. 8:597-613, 1997).A subject can receive a single injection, or, if additional injectionsare necessary, they can be repeated at six month intervals (or otherappropriate time intervals, as determined by the skilled practitioner)for an indefinite period and/or until the efficacy of the treatment hasbeen established.

Parenteral administration of the nucleic acid or vector of the presentinvention, if used, is generally characterized by injection. Injectablescan be prepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution of suspension in liquidprior to injection, or as emulsions. A more recently revised approachfor parenteral administration involves use of a slow release orsustained release system such that a constant dosage is maintained. See,e.g., U.S. Pat. No. 3,610,795, which is incorporated by referenceherein. For additional discussion of suitable formulations and variousroutes of administration of therapeutic compounds, see, e.g., Remington:The Science and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, MackPublishing Company, Easton, Pa. 1995.

7. Pharmaceutical Carriers/Delivery of Pharmaceutical Products

As described above, the compositions can also be administered in vivo ina pharmaceutically acceptable carrier. By “pharmaceutically acceptable”is meant a material that is not biologically or otherwise undesirable,i.e., the material can be administered to a subject, along with thenucleic acid or vector, without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.The carrier would naturally be selected to minimize any degradation ofthe active ingredient and to minimize any adverse side effects in thesubject, as would be well known to one of skill in the art.

The compositions can be administered orally, parenterally (e.g.,intravenously), by intramuscular injection, by intraperitonealinjection, transdermally, extracorporeally, topically or the like,although topical intranasal administration or administration by inhalantis typically preferred. As used herein, “topical intranasaladministration” means delivery of the compositions into the nose andnasal passages through one or both of the nares and can comprisedelivery by a spraying mechanism or droplet mechanism, or throughaerosolization of the nucleic acid or vector. The latter is effectivewhen a large number of animals is to be treated simultaneously.Administration of the compositions by inhalant can be through the noseor mouth via delivery by a spraying or droplet mechanism. Delivery canalso be directly to any area of the respiratory system (e.g., lungs) viaintubation. The exact amount of the compositions required will vary fromsubject to subject, depending on the species, age, weight and generalcondition of the subject, the severity of the allergic disorder beingtreated, the particular nucleic acid or vector used, its mode ofadministration and the like. Thus, it is not possible to specify anexact amount for every composition. However, an appropriate amount canbe determined by one of ordinary skill in the art using only routineexperimentation given the teachings herein.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein.

The materials can be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These can be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter, et al., BioconjugateChem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281,(1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, etal., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., CancerImmunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie,Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). Vehicles such as “stealth” and otherantibody conjugated liposomes (including lipid mediated drug targetingto colonic carcinoma), receptor mediated targeting of DNA through cellspecific ligands, lymphocyte directed tumor targeting, and highlyspecific therapeutic retroviral targeting of murine glioma cells invivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

a) Pharmaceutically Acceptable Carriers

The compositions, including antibodies, can be used therapeutically incombination with a pharmaceutically acceptable carrier.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

Pharmaceutical compositions can include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions can also includeone or more active ingredients such as antimicrobial agents,antiinflammatory agents, anesthetics, and the like.

The pharmaceutical composition can be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Administration can be topically (includingophthalmically, vaginally, rectally, intranasally), orally, byinhalation, or parenterally, for example by intravenous drip,subcutaneous, intraperitoneal or intramuscular injection. The disclosedantibodies can be administered intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives can also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration can include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Some of the compositions can be administered as a pharmaceuticallyacceptable acid- or base-addition salt, formed by reaction withinorganic acids such as hydrochloric acid, hydrobromic acid, perchloricacid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid,and organic acids such as formic acid, acetic acid, propionic acid,glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid,succinic acid, maleic acid, and fumaric acid, or by reaction with aninorganic base such as sodium hydroxide, ammonium hydroxide, potassiumhydroxide, and organic bases such as mono-, di-, trialkyl and arylamines and substituted ethanolamines.

b) Therapeutic Uses

The dosage ranges for the administration of the compositions are thoselarge enough to produce the desired effect in which the symptomsdisorder are effected. The dosage should not be so large as to causeadverse side effects, such as unwanted cross-reactions, anaphylacticreactions, and the like. Generally, the dosage will vary with the age,condition, sex and extent of the disease in the patient and can bedetermined by one of skill in the art. The dosage can be adjusted by theindividual physician in the event of any counterindications. Dosage canvary, and can be administered in one or more dose administrations daily,for one or several days.

8. Computer Readable Mediums

It is understood that the disclosed nucleic acids and proteins can berepresented as a sequence consisting of the nucleotides of amino acids.There are a variety of ways to display these sequences, for example thenucleotide guanosine can be represented by G or g. Likewise the aminoacid valine can be represented by Val or V. Those of skill in the artunderstand how to display and express any nucleic acid or proteinsequence in any of the variety of ways that exist, each of which isconsidered herein disclosed. Specifically contemplated herein is thedisplay of these sequences on computer readable mediums, such as,commercially available floppy disks, tapes, chips, hard drives, compactdisks, and video disks, or other computer readable mediums. Alsodisclosed are the binary code representations of the disclosedsequences. Those of skill in the art understand what computer readablemediums. Thus, computer readable mediums on which the nucleic acids orprotein sequences are recorded, stored, or saved.

Disclosed are computer readable mediums comprising the sequences andinformation regarding the sequences set forth herein. Also disclosed arecomputer readable mediums comprising the sequences and informationregarding the sequences set forth herein wherein the sequences do notinclude SEQ ID Nos: 37, 38, 39, 40, 41, and 42.

9. Compositions Identified by Screening with Disclosed Compositions

a) Computer Assisted Drug Design

The disclosed compositions can be used as targets for any molecularmodeling technique to identify either the structure of the disclosedcompositions or to identify potential or actual molecules, such as smallmolecules, which interact in a desired way with the disclosedcompositions. The nucleic acids, peptides, and related moleculesdisclosed herein can be used as targets in any molecular modelingprogram or approach.]

It is understood that when using the disclosed compositions in modelingtechniques, molecules, such as macromolecular molecules, will beidentified that have particular desired properties such as inhibition orstimulation or the target molecule's function. The molecules identifiedand isolated when using the disclosed compositions, such as CR2, DAF,CD59, CR1, MCP, Crry, IgG1, IgM, IgG3, CVF, CR2-DAF, DAF-CR2, CR2-CD59,CD59-CR2, CR2-CR1, CR1-CR2, CR2-MCP, MCP-CR2, CR2-Crry, Crry-CR2,CR2-IgG1 Fc (human), CR2-IgM Fc, CR2-IgG3 Fc (murine), or CR2-CVF arealso disclosed. Thus, the products produced using the molecular modelingapproaches that involve the disclosed compositions, such as, CR2, DAF,CD59, CR1, MCP, Crry, IgG1, IgM, IgG3, CVF, CR2-DAF, DAF-CR2, CR2-CD59,CD59-CR2, CR2-CR1, CR1-CR2, CR2-MCP, MCP-CR2, CR2-Crry, Crry-CR2,CR2-IgG1 Fc (human), CR2-IgM Fc, CR2-IgG3 Fc (murine), or CR2-CVF, arealso considered herein disclosed.

Thus, one way to isolate molecules that bind a molecule of choice isthrough rational design. This is achieved through structural informationand computer modeling. Computer modeling technology allows visualizationof the three-dimensional atomic structure of a selected molecule and therational design of new compounds that will interact with the molecule.The three-dimensional construct typically depends on data from x-raycrystallographic analyses or NMR imaging of the selected molecule. Themolecular dynamics require force field data. The computer graphicssystems enable prediction of how a new compound will link to the targetmolecule and allow experimental manipulation of the structures of thecompound and target molecule to perfect binding specificity. Predictionof what the molecule-compound interaction will be when small changes aremade in one or both requires molecular mechanics software andcomputationally intensive computers, usually coupled with user-friendly,menu-driven interfaces between the molecular design program and theuser.

Examples of molecular modeling systems are the CHARMm and QUANTAprograms, Polygen Corporation, Waltham, Mass. CHARMm performs the energyminimization and molecular dynamics functions. QUANTA performs theconstruction, graphic modeling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

A number of articles review computer modeling of drugs interactive withspecific proteins, such as Rotivinen, et al., 1988 Acta PharmaceuticaFennica 97, 159-166; Ripka, New Scientist 54-57 (Jun. 16, 1988);McKinaly and Rossmann, 1989 Annu. Rev. Pharmacol. Toxiciol. 29, 111-122;Perry and Davies, QSAR: Quantitative Structure-Activity Relationships inDrug Design pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989Proc. R. Soc. Lond. 236, 125-140 and 141-162; and, with respect to amodel enzyme for nucleic acid components, Askew, et al., 1989 J. Am.Chem. Soc. 111, 1082-1090. Other computer programs that screen andgraphically depict chemicals are available from companies such asBioDesign, Inc., Pasadena, Calif., Allelix, Inc, Mississauga, Ontario,Canada, and Hypercube, Inc., Cambridge, Ontario. Although these areprimarily designed for application to drugs specific to particularproteins, they can be adapted to design of molecules specificallyinteracting with specific regions of DNA or RNA, once that region isidentified.

Although described above with reference to design and generation ofcompounds which could alter binding, one could also screen libraries ofknown compounds, including natural products or synthetic chemicals, andbiologically active materials, including proteins, for compounds whichalter substrate binding or enzymatic activity.

10. Kits

Disclosed herein are kits that are drawn to reagents that can be used inpracticing the methods disclosed herein. The kits can include anyreagent or combination of reagent discussed herein or that would beunderstood to be required or beneficial in the practice of the disclosedmethods. For example, the kits could include primers to perform theamplification reactions discussed in certain embodiments of the methods,as well as the buffers and enzymes required to use the primers asintended. For example, disclosed is a kit for assessing a subject's riskfor cancer, asthma, systemic lupus erythematosus, rheumatoid arthritis,reactive arthritis, spndyarthritis, systemic vasculitis, insulindependent diabetes mellitus, multiple sclerosis, experimental allergicencephalomyelitis, Sjögren's syndrome, graft versus host disease,inflammatory bowel disease including Crohn's disease, ulcerativecolitis, Ischemia reperfusion injury, myocardial infarction, alzheimer'sdisease, transplant rejection (allogeneic and xenogeneic), thermaltrauma, any immune complex-induced inflammation, glomerulonephritis,myasthenia gravis, multiple sclerosis, cerebral lupus, Guillain-Barresyndrome, vasculitis, systemic sclerosis, anaphylaxis, catheterreactions, atheroma, infertility, thyroiditis, ARDS, post-bypasssyndrome, hemodialysis, juvenile rheumatoid, Behcets syndrome, hemolyticanemia, pemphigus, bullous pemphigoid, stroke, atherosclerosis, andscleroderma.

11. Compositions with Similar Functions

It is understood that the compositions disclosed herein have certainfunctions, such as modulating complement activity or binding CR2, CR3,or C3b. Disclosed herein are certain structural requirements forperforming the disclosed functions, and it is understood that there area variety of structures which can perform the same function which arerelated to the disclosed structures, and that these structures willultimately achieve the same result, for example stimulation orinhibition complement activity.

E. METHODS OF MAKING THE COMPOSITIONS

The compositions disclosed herein and the compositions necessary toperform the disclosed methods can be made using any method known tothose of skill in the art for that particular reagent or compound unlessotherwise specifically noted.

Disclosed are methods of making a composition comprising a construct,wherein the construct comprises CR2 and a modulator of complement. Alsodisclosed are methods of making a composition, wherein the compositionis the composition of the invention.

1. Peptide Synthesis

One method of producing the disclosed proteins, such as SEQ ID NO: 6, isto link two or more peptides or polypeptides together by proteinchemistry techniques. For example, peptides or polypeptides can bechemically synthesized using currently available laboratory equipmentusing either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc(tert-butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., FosterCity, Calif.). One skilled in the art can readily appreciate that apeptide or polypeptide corresponding to the disclosed proteins, forexample, can be synthesized by standard chemical reactions. For example,a peptide or polypeptide can be synthesized and not cleaved from itssynthesis resin whereas the other fragment of a peptide or protein canbe synthesized and subsequently cleaved from the resin, thereby exposinga terminal group which is functionally blocked on the other fragment. Bypeptide condensation reactions, these two fragments can be covalentlyjoined via a peptide bond at their carboxyl and amino termini,respectively, to form an antibody, or fragment thereof (Grant G A (1992)Synthetic Peptides: A User Guide. W.H. Freeman and Co., N.Y. (1992);Bodansky M and Trost B., Ed. (1993) Principles of Peptide Synthesis.Springer-Verlag Inc., NY (which is herein incorporated by reference atleast for material related to peptide synthesis). Alternatively, thepeptide or polypeptide is independently synthesized in vivo as describedherein. Once isolated, these independent peptides or polypeptides can belinked to form a peptide or fragment thereof via similar peptidecondensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segmentsallow relatively short peptide fragments to be joined to produce largerpeptide fragments, polypeptides or whole protein domains (Abrahmsen L etal., Biochemistry, 30:4151 (1991)). Alternatively, native chemicalligation of synthetic peptides can be utilized to syntheticallyconstruct large peptides or polypeptides from shorter peptide fragments.This method consists of a two step chemical reaction (Dawson et al.Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779(1994)). The first step is the chemoselective reaction of an unprotectedsynthetic peptide-thioester with another unprotected peptide segmentcontaining an amino-terminal Cys residue to give a thioester-linkedintermediate as the initial covalent product. Without a change in thereaction conditions, this intermediate undergoes spontaneous, rapidintramolecular reaction to form a native peptide bond at the ligationsite (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I etal., J. Biol. Chem., 269:16075 (1994); Clark-Lewis I et al.,Biochemistry, 30:3128 (1991); Rajarathnam K et al., Biochemistry33:6623-30 (1994)).

Alternatively, unprotected peptide segments are chemically linked wherethe bond formed between the peptide segments as a result of the chemicalligation is an unnatural (non-peptide) bond (Schnolzer, M et al.Science, 256:221 (1992)). This technique has been used to synthesizeanalogs of protein domains as well as large amounts of relatively pureproteins with full biological activity (deLisle Milton R C et al.,Techniques in Protein Chemistry IV. Academic Press, New York, pp.257-267 (1992)).

2. Process for Making the Compositions

Disclosed are processes for making the compositions as well as makingthe intermediates leading to the compositions. For example, disclosedare nucleic acids in SEQ ID NOs: 5. There are a variety of methods thatcan be used for making these compositions, such as synthetic chemicalmethods and standard molecular biology methods. It is understood thatthe methods of making these and the other disclosed compositions arespecifically disclosed.

Disclosed are nucleic acid molecules produced by the process comprisinglinking in an operative way a nucleic acid comprising the sequence setforth in SEQ ID NO: 25 and a sequence controlling the expression of thenucleic acid.

Also disclosed are nucleic acid molecules produced by the processcomprising linking in an operative way a nucleic acid moleculecomprising a sequence having 80% identity to a sequence set forth in SEQID NO: 25, and a sequence controlling the expression of the nucleicacid.

Disclosed are animals produced by the process of transfecting a cellwithin the animal with any of the nucleic acid molecules disclosedherein. Disclosed are animals produced by the process of transfecting acell within the animal any of the nucleic acid molecules disclosedherein, wherein the animal is a mammal. Also disclosed are animalsproduced by the process of transfecting a cell within the animal any ofthe nucleic acid molecules disclosed herein, wherein the mammal ismouse, rat, rabbit, cow, sheep, pig, or primate.

Also disclose are animals produced by the process of adding to theanimal any of the cells disclosed herein.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains. Thereferences disclosed are also individually and specifically incorporatedby reference herein for the material contained in them that is discussedin the sentence in which the reference is relied upon.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

F. EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the Dr.Tomlinson regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.), butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

1. Example 1 Complement Receptor 2 (CR2)-Mediated Targeting ofComplement Inhibitors to Sites of Complement Activation

a) Methods

(1) Cell Lines and DNA.

All DNA manipulations were carried out in the mammalian expressionvector PBM, derived from p118-mIgG1 (30) by deletion of mouse IgG1Fccoding region. Chinese hamster ovary (CHO) cells were used for proteinexpression and were maintained in Dulbecco's modified Eagle's medium(DMEM) (GIBCO Invitrogen Corp, Carlsbad, Calif.) supplemented with 10%FCS. Stably transfected CHO cell clones were cultivated in the presenceof G418, and for recombinant protein expression cells were cultured insuspension in CHO-S-SFM II without FCS (GIBCO). U937 cells were culturedin RPMI (GIBCO), 10% FCS.

(2) Antibodies, Reagents and Serum.

Rabbit antiserum to CHO cell membrane, purified human DAF and CD59 wasprepared by standard techniques (31). Mouse anti-DAF mAb 1H4 (32), ratanti-CD59 mAb YTH53.1 (33) and mouse anti human CR2 mAb 171 (binds toSCR 1-2) (34) are described. Anti-sheep erythrocyte IgM was fromResearch Diagnostic Inc. (Flanders, N.J.). All secondary antibodies werepurchased from Sigma (St. Louis, Mo.). Purified recombinant sCD59 was agift from Dr. B. P. Morgan (University of Wales, Cardiff, UK).C6-depleted human serum was purchased from Quidel (San Diego, Calif.)and normal human serum (NHS) was obtained from the blood of healthyvolunteers in the laboratory.

(3) Construction of Expression Plasmids and Protein Expression.

The recombinant fusion proteins and soluble complement inhibitorsprepared are depicted in FIG. 1. cDNA constructs were prepared byjoining the CR2 sequence encoding the 4 N-terminal SCR units (residues1-250 of mature protein, Swissprot accession no. P20023) to sequencesencoding extracellular regions of DAF or CD59. The complement inhibitorsequences used encoded residues 1-249 of mature DAF protein sequence(Swissprot accession no. P08174) and residues 1-77 of mature CD59protein sequence (Swissprot accession no. P13987). To join CR2 tocomplement inhibitor sequences, linking sequences encoding SS(GGGGS)₃and (GGGS)₂ were used for fusion proteins containing CR2 at theC-terminus and N-terminus, respectively. Gene constructs were preparedby standard PCR methodology (35). All cloning steps were performed inthe PBM vector that was also used for protein expression (30). Forexpression, plasmids were transfected into CHO cells using lipofectamineaccording to manufacturer's instructions (GIBCO). Stably transfectedclones were selected by limiting dilution as described (30) and proteinexpression of clones quantitated by ELISA.

(4) ELISA and Protein Assays.

Detection of recombinant proteins and determination of relative proteinconcentration in culture supernatants was achieved using a standardELISA technique (31). Depending on which type of recombinant protein wasbeing assayed, the capture antibody was either anti-DAF mAb 1H4 oranti-CD59 mAb YTH53.1. Primary detection antibodies were either anti-DAFor anti-CD59 rabbit polyclonal antibody. In some ELISAs, anti-CR2 mAbA-3 was also used as primary detection antibody, and although lesssensitive, similar data was obtained. The protein concentration ofrecombinant proteins was determined either by UV absorbance or by usinga BCA protein assay kit (Pierce Chemical Company, Rockford Ill.).

(5) Protein Purification.

Recombinant proteins were purified from culture supernatant by affinitychromatography. Affinity columns were prepared by coupling eitheranti-DAF 1H4 mAb or anti-CD59 YTH53.1 mAb to HiTrap NHS-activatedaffinity columns (Pharmacia Biotech, New Jersey, USA) as described bythe manufacturer. Culture supernatants containing recombinant proteinswere adjusted to pH 8.0 and applied to affinity columns at a flow rateof 0.5 ml/min. The column was washed with 6 to 8 column volumes of PBS,and recombinant proteins eluted with 2 to 3 column volumes of 0.1 Mglycine, pH 2.4. The fractions containing fusion protein were collectedinto tubes containing 1 M Tris buffer, pH 8.0 and dialyzed against PBS.

(6) SDS-PAGE and Western Blotting.

Purified recombinant proteins were separated in SDS-PAGE 10% acrylamidegels (Bio-Rad Life Science, Hercules, Calif.) under nonreducingconditions. Gels were stained with Coomassie blue. For Western blotting,standard procedures were followed (31). Briefly, separated proteins weretransferred to a polyvinylidene fluoride membrane, and the transferredproteins detected by means of either anti-DAF mAb 1H4 or anti-CD59 mAbYTH53.1. Membranes were developed with ECL detection kit (AmershamBiosciences, Piscataway, N.J.). CR2-CD59 was also analyzed by SDS-PAGEfollowing glycanase treatment. CR2-CD59 (2 mg) was heated at 95° C. for3 min in 15 mM sodium phosphate buffer (pH 7.5) containing 0.1% SDS, 10mM 2-mercaptoethanol and 5 mM EDTA. After cooling, CR2-CD59 wasincubated with 3 U of Flavobacterium meningosepticum N-glycanase (EC3.5.1.52, Sigma) for 20 h at 37° C. in the presence of 1% Nonidet P40and 0.3 mM PMSF.

(7) Flow Cytometry.

Binding of recombinant fusion proteins to C3-opsonized cells wasdetermined by flow cytometry. CHO cells were incubated in 10% anti-CHOantiserum (30 min/4° C.), washed and incubated in 10% C6-depleted NHS(45 min/37° C.). The C3 opsonized cells were then washed and incubatedwith 1 μM recombinant protein (60 min/4° C.). After washing, cells wereincubated with 10 μg/ml of either anti-DAF mAb 1H4 or anti-CD59 mAbYTH53.1 as appropriate (30 min/4° C.), followed by FITC-conjugatedsecondary antibody (1:100, 30 min/4° C.). Cells were then washed, fixedwith 2% paraformaldehyde in PBS, and analyzed using a FACScan flowcytometer (Becton Dickinson Immunocytometry Systems, San Jose, Calif.).All incubations and washes were performed in DMEM.

(8) Analysis of CR2 Fusion Protein Binding to C3 Ligand.

Kinetic analysis of the interaction of the CR2 fusion proteins withC3dg-biotin was performed using surface plasmon resonance (SPR)measurements made on a BIAcore 3000 instrument. Human C3dg-biotin,prepared as described (36), was bound to the surface of BIAcorestreptavidin (SA) sensor chips by injecting C3dg-biotin at 50 μg/ml overthe surface of one flow cell of the chip at 2 μl/minute for 20 minutes.The flow buffer was 0.5×PBS+0.05% Tween 20. The SPR signal from capturedC3dg generated BIAcore response units ranging from 250-500. Controlstreptavidin-coated flow cells were run in the absence of protein.Binding was evaluated over a range of CR2 fusion protein concentrations(15.6-500 nM) in 0.5×PBS, 0.05% Tween 20 at 25° C. at a flow rate of 25μl/minute. CR2 fusion protein samples were injected in 50 μl aliquotsusing the kinject command. Association of the fusion proteins with theligand was monitored for 120 seconds, after which the complex wasallowed to dissociate in the presence of buffer only for an additional120 seconds. The binding surface was regenerated between analyses ofdifferent fusion protein concentrations by a 10 second pulse of 200 mMsodium carbonate (pH 9.5) at 50 μl/min. Binding of CR2 fusion proteinfragments to C3d-immobilized flow cells was corrected for binding tocontrol flow cells. Binding data were fitted to a 1:1 Langmuir bindingmodel using BIAevaluation Version 3.1 software (BIAcore) and evaluatedfor best fit by low residual and χ² values. The kinetic dissociationprofiles obtained were used to calculate on and off-rates (k_(a) andk_(d)) and affinity constants (K_(D)) using the BLAevaluation Version3.1 program. Between experiments, the streptavidin surface wasregenerated with a 60-s pulse of 50 mM sodium hydroxide (pH 9.5) at 50μl/minute, and C3dg-biotin was reapplied as described above.

(9) Complement Lysis Assays.

CHO cells at 60%80% confluence were detached with versene (GIBCO),washed twice, and resuspended to 10⁶/ml in DMEM. Cells were sensitizedto complement by adding 10% rabbit anti-CHO cell membrane antiserum tocells (30 min/4° C.). Antiserum was then removed and cells resuspendedin NHS diluted in DMEM. Final assay volumes were either 50 or 100 μl.After 45 min at 37° C., cell viability was determined either bytrypanblue exclusion (both live and dead cells counted) or ⁵¹Cr release (37).Both assays gave similar results. To assay complement inhibitoryactivity of recombinant proteins, the proteins were diluted in DMEM andadded to NHS before addition to CHO cells. A final concentration of 10%NHS was used which resulted in approximately 90% lysis of unprotectedantibody sensitized CHO cells. Inhibition of complement-mediatedhemolysis was determined using antibody-sensitized sheep erythrocytes(EA) (Advanced Research Technologies, San Diego, Calif.). Hemolyticassays were carried out in gelatin veronal buffer (GVB⁺⁺) (AdvancedResearch Technologies) in a final volume of 300 μl containing 2.5×10⁷EA, NHS at a final dilution of 1/300 and incremental concentrations offusion protein. Reaction mixtures were incubated at 37° C. for 60 minand reactions were stopped by addition of 300 μl PBS containing 10 mMEDTA. Cells were removed by centrifugation and cell lysis assayed byspectrophotometric quantitation of hemoglobin in the supernatant at 413mm.

(10) Adhesion of U937 Cells to Erythrocytes.

Assays of CR3-dependent adhesion to C3-opsonized erythrocytes wereperformed essentially as described (38). Briefly, fresh sheeperythrocytes (SRBC) were sensitized with a pre-determinedsub-agglutinating amount of rabbit anti-SRBC IgM for 30 min at 37° C. inGVB (Advanced Research Technologies). After washing twice, C3b-opsonizedSRBC were prepared by incubating IgM-sensitized SRBC with an equalvolume of a 1:2 dilution of C6-deficient human serum in GVB (120 min/37°C.). Cells were washed twice and pellets resuspended in GVB. Themajority of C3 bound to erythrocytes following this treatment is in theform of iC3b or C3d degredation products (CR2 ligands) due to the shorthalf life of C3b in serum. U937 cells (4×10⁵ cells in 200 μl) were addedto 50 μl of C3 opsonized SRBC (2×10⁶ cells) and the mixture centrifuged(4 min/40×g) and left at room temperature for 90 min. Cells were thenexamined by phase contrast microscopy and number of U937 cells adherentto erythrocytes determined. At least 100 erythrocytes were scored persample, and average number of U937 cells bound per erythrocytecalculated. Triplicate determinations were made for each experimentperformed. In some experiments, U937 cells were cultured for 3 days inthe presence of 50 ng/ml phorbol myristate acetate (PMA) before harvest,a treatment that results in upregulation of CR3 (39, 40). Cellsincubated with IgM-coated SRBC alone, or SRBC incubated directly withC6-deficient human serum were used as controls.

(11) Biodistribution Studies.

Standard procedures for determining tissue distribution of injectedradiolabeled proteins were followed (41, 42). Briefly, 1.7 μg of¹²⁵I-labeled CR2-DAF (4.20×10⁶ cpm/mg) or sDAF (4.84×10⁶ cpm/mg) wereinjected into the tail vein of 34 week old female NZB/NZW F1 mice(Jackson Labs, Bar Harbor, Me.). After 24 h, a blood sample was takenand major organs were removed, shredded and washed in PBS containing 10mM EDTA, weighed and counted. Targeting specificity was evaluated aspercent injected dose per gram tissue. Proteins were iodinated usingiodogen method according to manufacturers instructions (Pierce ChemicalCo.).

(12) Immunofluorescence Microscopy.

CR2-DAF or sDAF (270 μg) was injected into the tail vein of 24-week-oldMRL/lpr mice. Twenty-four hours later, kidneys were removed and snapfrozen. Cryostat sections (5 μm) prepared from frozen kidneys were fixedin acetone and processed for indirect immunofluorescence microscopy. Anequimolar mixture of mouse antihuman DAF 1A10 and 1H6 mAbs were used asprimary detection antibodies (final concentration, 10 μg/ml) with ananti-mouse IgG Fc-specific FITC-conjugated secondary antibody (F4143,Sigma-Aldrich). Standard procedures were followed (49, incorporatedherein by reference for its teaching of antibody staining techniques),except that to reduce background staining, most likely caused bydeposited immune complexes in the mouse kidney, the secondaryFITC-labeled antibody was diluted 1:800 (10 times the recommendeddilution). Digital images were acquired and optimized with AdobePhotoshop using identical settings.

b) Results

(1) Construct Design, Expression and Purification.

Recombinant fusion proteins contained the four N-terminal SCR units ofhuman CR2 linked to either the N or C terminus of soluble forms of humanCD59 or DAF (constructs depicted in FIG. 1). Recombinant proteins werepurified from the culture supernatant of stably transfected CHO cellclones with yields of between 100-200 μg/l. Analysis of purifiedrecombinant proteins by SDS-PAGE and Western blot revealed proteinswithin expected molecular weight range (FIG. 2), and except forCR2-CD59, all proteins migrated as a single band. The two bands seen forCR2-CD59 were due to differences in glycosylation, since CR2-CD59migrated as a single band following glycanase treatment.

(2) Targeting of Fusion Proteins to Complement Opsonized Cells.

C3 ligand for CR2 was deposited on CHO cells by incubation of CHO cellswith complement activating antibody and C6-depleted serum (to preventMAC formation and cell lysis). All CR2-containing fusion proteins, butnot sCD59 or sDAF, bound to C3-coated CHO cells (FIG. 3).

(3) Kinetic Analysis of Interaction Between Fusion Proteins and C3dgLigand.

A comparison of the affinity of the different recombinant fusionproteins for the CR2 ligand C3dg was determined by surface plasmonresonance measurements. The experiments were performed by passingvarying concentrations of the fusion proteins over Biacore streptavidinchips containing captured C3dg-biotin (approximately 2000 responseunits). Kinetic analysis of the data showed the best fit to a 1:1(Langmuir) binding interaction model using global fitting parameters(FIG. 4). Both of the fusion proteins with CR2 at the N-terminus(CR2-DAF and CR2-CD59) showed similar binding profiles, with a fastassociation and a fast dissociation rate. In contrast, binding of fusionproteins with CR2 at the C-terminus (DAF-CR2 and CD59-CR2) showed slowassociation and dissociation rates (FIG. 4, Table 1). The N-terminus CR2fusion proteins, however, bound with the highest affinity (Table 1).CD59 fusion proteins bound with a higher affinity than DAF fusionproteins. Soluble DAF and sCD59 did not bind to immobilized C3dg.

(4) Complement Inhibitory Activity of Fusion Proteins.

Complement inhibitory activity of the targeted and untargeted complementinhibitors was analyzed by measuring their effect on complement-mediatedlysis of both CHO cells and erythrocytes. In these experiments, antibodysensitized cells and recombinant proteins were incubated in human serumat a concentration that resulted in 90-100% lysis of unprotected cells.For both cell types, the targeted complement inhibitors weresignificantly more effective than their respective untargeted proteinsat inhibiting complement-mediated lysis. Targeted DAF proteins were moreeffective inhibitors than targeted CD59 (FIGS. 5 and 6). Fusion proteinscontaining CR2 linked to the N-terminus of either DAF and CD59 were moreeffective inhibitors than C-terminal CR2 fusion proteins. The mostpotent inhibitor of complement lysis was CR2-DAF, requiring aconcentration of 18 nM for 50% inhibition of CHO cell lysis. Incontrast, untargeted sDAF required a concentration of 375 nM for 50%inhibition of CHO cell lysis, a 20-fold difference (FIG. 5 a). sCD59 wasa particularly poor inhibitor of complement and provided only 25%protection from CHO cell lysis at 500 nM, the highest concentrationtested. CR2-CD59, however, provided 50% inhibition of CHO cell lysis at102 nM and was more effective than untargeted sDAF (FIG. 6 a). Table 4compares the inhibitory activities of the different recombinantcomplement inhibitors. The higher complement inhibitory activity of theN-terminus CR2 fusion proteins correlated with the higher affinity theseproteins exhibited for C3dg ligand (Table 3).

There were some differences between the relative effectiveness of thecomplement inhibitors at protecting CHO cells and erythrocytes formcomplement-mediated lysis. This was particularly true for the DAFinhibitors; sDAF was significantly more effective at protectingerythrocytes than CHO cells from complement, although targeted DAF wasstill more effective. There was also little difference in the inhibitoryactivity of CR2-DAF and DAF-CR2 when erythrocytes were the target cellsfor complement lysis.

(5) Effect of CR2-Fusion Proteins on Cell Adhesion.

Complement receptor 3 is a leukocyte receptor involved in endothelialadhesion and diapedesis and the activation of cell cytolytic mechanisms(phagocytosis and degranulation). Since CR2 and CR3 share the same iC3bcomplement ligand, it was determined whether CR2 fusion proteinsinterfered with CR3-mediated cell binding. For these experiments U937, awell characterized promonocytic cell line (CR2⁻, CR3⁺) that binds toiC3b coated erythrocytes in a CR3-dependent mechanism, was used (40).All of the CR2 fusion proteins, but not sDAF or sCD59, significantlyinhibited the binding of U937 cells to C3 opsonized sheep erythrocytes(P<0.01). Each CR2 fusion protein inhibited U937 binding to a similarextent at a concentration of 500 mM (FIG. 7). Similar data was obtainedin an experiment using U937 cells that were stimulated with PMA, atreatment that results in upregulation of CR3 (39, 40). For complementopsonization of erythrocytes, IgM was used to activate complement sinceIgG deposited on the erythrocytes would engage Fcγ receptors expressedon U937 cells. U937 cells also express CR4 (p150,95, CD11c/CD18), athird receptor sharing the iC3b ligand. However, binding of U937 cellsto C3-opsonized erythrocytes is CR4-independent, probably due to theassociation of CR4 with the cytoskeleton and its immobility in themembrane (40).

(6) Targeting of CR2-DAF to the Kidneys of Nephritic Mice.

To determine whether a CR2 fusion protein will target a site ofcomplement activation and disease in vivo, a biodistribution study ofCR2-DAF and sDAF in female NZB/W F1 mice was performed. NZB/W F1 micedevelop a spontaneous autoimmune disease that is very similar to humansystemic lupus erythematosus (SLE), with the production ofautoantibodies and the development of severe immune complex-mediatedglomerulonephritis that is associated with complement deposition from 26to 28 weeks of age (4, 52). Biodistribution of [¹²⁵I]CR2-DAF and[¹²⁵]sDAF in 34-week-old NZB/W F1 mice was determined at 24 hours and 48hours after injection. Twenty-four hours after tail-vein injection of[¹²⁵I]CR2-DAF, a significantly higher proportion of radioactivity waslocalized to the kidney than to the other organs that were examined(FIG. 25 a). At 48 hours after injection of [¹²⁵I]CR2-DAF, there was asimilar level of radioactivity in the kidney as at 24 hours, butradioactivity in the liver and spleen was increased and bloodradioactivity decreased (FIG. 25 b). The liver and spleen are sites ofimmune complex clearance and likely account for increased targeting of[¹²⁵I]CR2-DAF to these organs at the later time point. [¹²⁵I]sDAF showedno preferential binding in the kidney or any other organ (FIG. 25, a andb). In 8-weekold prenephritic NZB/W F1 mice, there was no evidence of[¹²⁵I]CR2-DAF targeting to the kidney (FIG. 25 c). Of further interest,[¹²⁵I]sDAF was cleared much more rapidly from the circulation than[¹²⁵I]CR2-DAF, suggesting that the CR2 moiety is functioning to prolongthe circulatory half-life of the fusion protein. However, the level of[¹²⁵I]CR2-DAF in the blood of younger mice at 24 hours was about halfthat recorded in the older mice, and the long circulatory half-life of[¹²⁵I]CR2-DAF may be a consequence, at least in part, of it binding tocirculating immune complexes.

Targeting of CR2-DAF to complement deposited in the kidney was alsoexamined in another murine model of SLE by direct examination of kidneysections. Similar to female NZB/W F1 mice, MRL/lpr mice develop severeproliferative glomerulonephritis with the deposition of complement inassociation with glomerular immune deposits by 24 weeks of age (53,incorporated herein for its teaching of this mouse model). CR2-DAF andsDAF were injected into the tail vein of 24-week-old MRL/lpr mice, andkidney sections were analyzed 24 hours later for human DAFimmunoreactivity by fluorescence microscopy. Kidney sections from amouse injected with CR2-DAF displayed a high level of DAF staining, withpreferential localization in glomeruli in a pattern identical to thatseen for immune complexes. No DAF staining was evident in glomeruli froma mouse injected with sDAF (FIG. 26).

c) Conclusions

This study describes the generation and characterization of solublehuman DAF and CD59 containing proteins that are targeted to a site ofcomplement activation. The targeted proteins were significantly morepotent at inhibiting complement than their untargeted counterparts.Targeting of CD59 and DAF was achieved by linking the inhibitors to afragment of human CR2 that binds complement C3 activation products. TheC3 ligands for CR2 are relatively long lived and are covalently bound,often in large quantities, at sites of complement activation. Thus,CR2-mediated targeting of complement inhibition is of therapeuticbenefit for numerous complement-associated diseases or disease states.Consistent with this hypothesis, CR2-DAF was shown to target to thekidneys of nephritic NZB/W F1 mice. These mice produce autoantibodieswith consequent formation and deposition of immune complexes in thekidney resulting in complement activation and deposition (2, 43). HumanCR2 binds human and mouse C3 ligands with similar affinities (44), andthe biodistribution studies establish that a CR2-fusion protein retainstargeting function in vivo. This study establishes the feasibility ofthis approach for human complement inhibition. The targeting approachcan also be effective for other inhibitors of complement activation suchas soluble CR1, which is in clinical trials and is a more potentinhibitor of complement than DAF in vitro (9).

The relative affinities for C3dg of the different CR2 fusion proteins isreminiscent of the affinities of SCR 1-2 of CR2 and SCR 1-15 of CR2 forC3dg. The KD values for CR2 SCR1-2 and CR2 SCR 1-15 interactions withC3dg were similar, but CR2 SCR 1-2 associated and dissociated muchfaster, indicating a contribution of the additional SCR domains tooverall affinity (36). Analysis of the solution structure of another SCRcontaining protein, factor H, indicated that SCR domains are folded backon themselves and interactions between SCR domains can modulate C3ligand binding characteristics (45). Conformational variability betweenSCR domains is predicted to result from different (native) linkerlengths, with longer linkers providing greater conformationalflexibility. In this context, the CR2 and DAF SCR domains are linkedwith a relatively long ser-gly linker, and this can permit the fusionpartners to fold back on one another resulting in SCR-SCR interactionsthat can modulate CR2 binding affinity.

Complement-mediated lysis assays were performed using antibodysensitized CHO cells or sheep erythrocytes as targets. There were markeddifferences in the relative activities of some of the complementinhibitors at protecting the different cells from complement-mediatedlysis. sDAF, DAF-CR2, and CD59-CR2 were significantly more effective atprotecting sheep erythrocytes than CHO cells from complement-mediatedlysis. Unlike rythrocytes, complement-mediated lysis of nucleated cellsis not due entirely to colloid osmotic deregulation, and the depositionof multiple MACs in the plasma membrane is required (46-48). Themajority of previous studies investigating the inhibitory activity ofsoluble (untargeted) complement inhibitors have been performed usingerythrocytes as target cells for complement mediated lysis. However, CHOcells likely represent a more physiologically relevant target for invitro experiments.

Different mechanisms of complement-mediated damage are implicated indifferent disease conditions and different diseases can benefit frominhibition strategies acting at different points in the pathway. Forexample, if applicable for the disease, a particular benefit of blockingcomplement at a late step in the pathway would be that host defensefunctions and immune homeostasis mechanisms of complement would remainintact. Thus, a CD59-based inhibitor would provide advantages overinhibitors of complement activation in diseases in which the terminalcytolytic pathway is primarily implicated in pathogenesis. Soluble CD59is unlikely to have therapeutic benefit due to its very poor activity invitro, but it was shown herein that CR2-mediated targeting of CD59significantly increased its complement inhibitory activity. In fact,CR2-CD59 was more effective at inhibiting complement-mediated lysis thansDAF, and sDAF has shown therapeutic efficacy in vivo (8). Rodentanalogues of CR2-CD59 can also be a useful tools for dissecting therelative roles of early complement activation products vs. MAC formationin disease pathogenesis. The relative contributions of the differentcomplement activation products to tissue injury in many disease statesis poorly understood and controversial.

The CR2 fusion proteins inhibited the binding of U937 cells toC3-opsonized erythrocytes. CR2 and CR3 both bind iC3b, and this dataindicates that CR2 fusion proteins act as CR3 antagonists since U937binding to C3-opsonized erythrocytes is CR3-dependent (40). As anadhesion molecule, CR3 mediates endothelial adhesion and diapedesis atsites of inflammation via its high affinity interaction withintercellular adhesion molecule-1 (ICAM-1). As a complement receptor,CR3 promotes and enhances phagocytosis and degranulation via itsinteraction with iC3b. Both ICAM-1 and iC3b bind to overlapping epitopeson CR3 (see Ross review). CR3 can thus be an important determinant inpromoting cell-mediated tissue damage at sites of inflammation, andantibodies that block CR3 have shown effectiveness in severalinflammatory conditions (see Ross review). The antagonistic effect ofCR2 on CR3 binding therefore indicates a second anti-inflammatorymechanism of action of the CR2-complement inhibitor fusion proteins thatact synergistically with complement inhibition.

Targeting complement inhibitors to sites of complement activation anddisease can considerably enhance their efficacy. Indeed, for diseasestates that would benefit from CD59-based therapy, the targeting of CD59to the site of complement activation will be a requirement. An advantageof CR2-mediated targeting over other targeting approaches, such asantibody-mediated targeting, is that the CR2 moiety targets anyaccessible site of complement activation and has broad therapeuticapplication. CR2 fusion proteins can also act as CR3 antagonists, andthis can represent a second important therapeutic benefit. HumanCR2-complement inhibitor fusion proteins are also much less likely to beimmunogenic than recombinant inhibitors containing antibody variableregions. The predicted ability of targeted inhibitors of complementactivation to provide an effective local concentration with low levelsof systemic inhibition also diminishes the possibility of compromisinghost defense mechanisms, particularly with long term systemic complementinhibition (this is a less important consideration for CD59-basedinhibitors). CR2-targeted inhibitors can also target infectious agentsthat activate complement.

2. Example 2 Targeted Complement Inhibition and Activation

a) Complement Inhibitors (Inflammation/Bioincompatibility)

Complement inhibitors hold considerable promise for the therapy of manyautoimmune and inflammatory diseases, and disease states associated withbioincompatibility. A safe and effective pharmaceutical inhibitor ofcomplement is not currently available. Research has largely focused ondeveloping soluble inhibitors based on host membrane-boundcomplement-regulatory proteins. Recombinant forms of soluble CR1, MCP,DAF and Crry have been produced by removal of membrane-linking regions,and all proteins have been shown to be effective at reducinginflammation and complement-mediated tissue damage in various models ofdisease. Soluble CR1 and an antibody that blocks the function ofcomplement protein C5 are in clinical trials. There are, however,serious questions concerning the clinical use of systemicallyadministered soluble complement inhibitors. Complement plays a crucialrole in both innate and adaptive immunity, and the generation of C3b iscritical for the opsonization and leukocyte-mediated clearance of manypathogenic microorganisms. In addition, the fluid phase complementactivation product C5a has been shown to be important in controllinginfection and can be important in the clearance of pathogenic substancesfrom the circulation. Systemic inhibition of complement is thereforelikely to have serious consequences for the host regarding its abilityto control infection. Complement is also crucial for the effectivecatabolism of immune complexes, and this is a particularly importantconsideration in the use of complement inhibitors for the treatment ofautoimmune and immune complex diseases.

The targeting of complement inhibitors to sites of complement activationand disease can allow a much lower effective serum concentration andsignificantly reduce the level of systemic complement inhibition.Increased efficacy is an important benefit of targeted complementinhibitors, and targeting can also address the problem of a short halflife of soluble recombinant complement inhibitors in the circulation.

In addition to the above considerations with regard to the targeting ofcomplement inhibitors, selectively blocking different parts of thecomplement pathway can allow the generation of beneficial complementactivation products, but inhibit the generation of complement activationproducts involved in disease pathogenesis. For example, inhibitors ofcomplement activation (such as CR1, DAF, Crry) inhibit C3b, C5a andC5b-9 generation. Antibodies to C5 inhibit C5a and C5b-9 generation. Onthe other hand, CD59-based inhibitors do not effect C3b and C5ageneration, but block only C5b-9 formation (see FIG. 8). The terminalcomplement pathway and C5b-9 generation has been shown to be importantin promoting inflammation and is in particular implicated in theprogression of some diseases of the kidney (such as immune complexglomerulonephritis). Thus, for certain diseases, a CD59-based inhibitorcan inhibit disease pathogenesis without interfering with the generationof early complement activation products that are important for hostdefense and immune complex clearance. However, soluble CD59 is not aneffective inhibitor of complement (unlike inhibitors of activation DAF,CR1, MCP or Crry), and is unlikely to have any clinical application.Cell targeted CD59, however, can represent a viable therapeutic. Datausing antibody-mediated targeting of CD59 [Zhang et al., 1999, J. Clin.Invest., 103, 55-61], and the data presented herein with CR2-mediatedtargeting of CD59 show that CD59 targeted to a cell membrane issignificantly more effective than soluble untargeted CD59.

b) Complement Activators (Cancer)

The initial promise of anti-tumor complement activating monoclonalantibodies as cancer immunotherapeutic agents has not been realized. Onereason for this is the expression of complement inhibitory proteins ontumor cells (complement inhibitors are often upregulated on tumorcells). Thus, although certain antibodies have been shown to targettumors in humans and to activate complement on the tumor cell surface,the tumor cells resist complement-mediated destruction. There is a largebody of evidence from in vitro studies indicating an important role forcomplement inhibitors in tumor resistance to antibody therapy. Inaddition, reports [Caragine, et al., 2002, Cancer Res, 62, 1110-15;Chenet al., 2000, Cancer Res., 60, 3013-18; Baranyi et al., 1994,Immunology, 82, 522-8] have established that complement inhibitorsexpressed on the surface of tumor cells in vivo have functionalconsequences with regard to complement deposition and tumorigenesis.Enhancing complement deposition on tumor cells allows more effectiveimmune-mediated clearance of tumor cells and improve prospects forsuccessful immunotherapy using complement-activating anti-tumorantibodies. Enhanced complement activation overwhelms tumor cellexpressed complement inhibitory proteins.

c) Results-1

(1) Targeted Complement Inhibitor Fusion Protein

Examples of human fusion proteins that have been expressed, purified andcharacterized for targeting and assessed for complement inhibitoryfunction in vitro as previously described include the following:CR2-DAF, CR2-CD59, DAF-CR2, and CD59-CR2. The nucleotide sequences andpredicted amino acid sequences of mature human fusion proteins are shownin FIGS. 8-11.

(2) Expression and Purification

cDNA plasmid constructs encoding the fusion proteins were transfectedinto CHO cells and stably expressing clones isolated. Clones expressinghighest levels of fusion protein were selected. The selected clones weregrown in bioreactors and fusion proteins isolated from culturesupernatant by affinity chromatography. Affinity columns were preparedusing anti-DAF and anti-CD59 antibodies conjugated to sepharose.Recombinant proteins were analyzed by SDS-PAGE and Western blot (FIG.2).

(3) Binding of Fusion Proteins to C3 Ligands.

(a) Flow Cytometry

Flow cytometry experiments were conducted as previously described. Allof the CR2 containing fusion proteins bound to C3-coated CHO cells, asanalyzed by flow cytometry (FIG. 12). sDAF and sCD59 did not bind toC3-coated CHO cells.

(b) ELISA

ELISA experiments were conducted as previously described. In ELISAexperiments, CR2-containing constructs were added to wells coated withpurified C3dg. Binding was detected by means of anti-complementinhibitor antibodies and enzyme-conjugated secondary antibodies. All CR2containing constructs, but not sCD59 and sDAF bound to C3d.

(c) Surface Plasmon Resonance

Biotinylated C3dg (CR2 ligand) was bound to streptavidin coated BIAcorechips and binding kinetics of CR2 containing fusion proteins measured(FIGS. 13-16). sDAF and sCD59 did not bind to captured C3dg. Fusionproteins with CR2 at N-terminus bound with highest affinity. CD59containing fusion proteins bound with higher affinity than correspondingDAF containing fusion proteins.

(4) Complement Inhibitory Function of Fusion Proteins

The functional activity of the fusion proteins and soluble untargetedcomplement inhibitors was analyzed by measuring the effect of theproteins on complement-mediated cell lysis. Assays using Chinese hamsterovary (CHO) cells (FIGS. 17 and 18) and sheep erythrocytes (E) (FIGS. 19and 20) were used.

The targeted complement inhibitors provided significantly moreprotection from complement-mediated than soluble untargeted complementinhibitors. Fusion proteins containing CR2 at the N-terminus were themost effective for both DAF and CD59 containing fusion proteins.N-terminal CR2 fusion proteins also bound C3d with a higher affinitythan C-terminal CR2 fusion proteins in BIAcore experiments (see above).CR2-DAF was significantly more effective than CR2-CD59 at providingprotection from complement-mediated lysis in these assays. Of note,however, untargeted sCD59 possesses very weak complement inhibitoryactivity even at high concentration (unlike sDAF), and the targeting ofCD59 to the cell surface is a requirement for CD59 function.

The relative effectiveness of targeted vs. untargeted complementinhibitors for CHO cells and E is different. However, erythrocytes arelysed by “one hit” (ie. formation of a single MAC causes E lysis),whereas nucleated cells (such as CHO) posses addition resistancemechanisms (such as capping and shedding of MACs) and require depositionof multiple MACs for lysis. These differences likely account fordifferences in lysis inhibition data, and CHO cells likely represent themore physiologically relevant target for these in vitro experiments.

D) RESULTS-2

(1) Targeted Complement Activating Fusion/Conjugated Proteins

Human CR2-IgG1 Fc has been expressed and purified and shown toappropriately target C3 opsonized cells in vitro. Expression plasmidscontaining encoding sequences for human and mouse sCR2 (for conjugationwith CVF) and mouse fusion proteins have been prepared. Nucleotidesequence and predicted amino acid sequences of mature human fusionproteins are shown in FIG. 21

(2) Expression and Purification

cDNA encoding the first 4 SCRs of CR2 was linked to genomic sequenceencoding human IgG1 Fc region. Plasmid encoding the fusion protein wastransfected into CHO cells and stably expressing clones isolated. Clonesexpressing highest levels of fusion protein were selected. The selectedclone was grown in a bioreactor and fusion proteins isolated fromculture supernatant by protein A affinity chromatography. Recombinantprotein was analyzed by SDS-PAGE (FIG. 22) and Western blot. Proteinmigrated at expected molecular weight under reducing and nonreducingconditions (CR2-Fc is disulfide linked dimer). A murine plasmidconstruct encoding CR2-mouse IgG3 has been constructed.

(3) Binding of Fusion Proteins to C3 Ligands.

(a) Flow Cytometry

CR2-Fc bound to C3-coated CHO cells, as analyzed by flow cytometry (FIG.23).

(b) ELISA

In ELISA experiments, CR2-Fc was added to wells coated with purifiedC3dg. Binding was detected by means of anti-human Fc antibodies andenzyme-conjugated secondary antibodies. CR2-Fc bound to C3d.

(c) Surface Plasmon Resonance

Biotinylated C3dg (CR2 ligand) was bound to streptavidin coated BIAcorechips and binding of CR2-Fc demonstrated (FIG. 24).

3. Example 3 a) Antibody Targeted Complement Inhibitors in a Rat Modelof Acute Tubulointerstitial Injury

A panel of well characterized mouse anti-rat kidney monoclonalantibodies was used (49, 50, incorporated herein by reference for theirteaching regarding these antibodies and their sequences). The variableregion DNA from a total of 5 antibodies was isolated by standard PCRtechniques (35, incorporated herein by reference for its teachingsregarding PCR). All were successfully cloned and some were expressed assingle chain antibodies. All single chain antibodies recognized either arat kidney epithelial or endothelial cell line in vitro. One of themAbs, K9/9, binds to a glycoprotein identified on the epithelial cellsurface, and has specificity for the glomerular capillary wall andproximal tubules in vivo (49). This antibody was chosen as a targetingvehicle for investigation of targeted Crry- and CD59-mediated complementinhibition in a rat model of acute tubulointerstitial injury. Althoughthe K9/9 mAb was shown to induce glomerular damage in a previous study(49), the antibody was only pathogenic when administered together withFreunds adjuvant. In fact the pathogenic nature of K9/9 mAb (withadjuvant) was not reproduced.

There is a link between proteinuria and progressive renal damage andthere is data to support the hypothesis that proteinuria itself resultsin interstitial fibrosis and inflammation. The mechanism by whichproteinuria leads to nephrotoxic injury is not known, but there isevidence that complement plays a key role and that the MAC is theprincipal mediator of tubulointerstitial injury due to proteinuria. (Therole of complement and proteinuria in tubulointerstitial injury has beenrecently reviewed (51, 52)). Previous characterization of K9/9 mAb (seeabove (49)) suggested that the mAb targets appropriately for aninvestigation into the therapeutic use of targeted complement inhibitorsin a rat model tubulointerstitial injury induced by proteinuria. Theavailability of an inhibitor that can specifically block MAC formationwould allows an assessment of the role of MAC in tubulointerstitialinjury under clinically relevant conditions.

Plasmid constructs encoding single chain K9/9 antibody linked to ratCrry or rat CD59 were prepared (depicted in FIG. 27). Constructsexpressing soluble rat Crry (sCrry) and single chain K9/9 (targetingvehicle only) were also prepared. All recombinant proteins wereexpressed into the culture medium at over 15 mg/liter by Pichiafermentation in a 15 liter New Brunswick fermentor.

Recombinant proteins were characterized for targeting and complementinhibitory activity in vitro as described above using a rat epithelialcell line as target cells. Single chain K9/9, K9/9-Crry and K9/9-CD59specifically bound to rat epithelia cells in vitro. sCrry and K9/9-Crryinhibited complement deposition and lysis, and K9/9-CD59 inhibitedcomplement-mediated lysis. Both targeted complement inhibitors, sCrryand K9/9 single chain Ab were characterized in the rat puromycinaminonucleoside (PAN) nephrosis model (53, incorporated herein byreference for its teaching of sCrry and K9/9) (sCD59 was not evaluatedsince untargeted CD59 has only very poor complement inhibitory activity(54)). First, to confirm kidney targeting of K9/9 fusion proteins, invivo binding specificity was determined by biodistribution of iodinatedproteins as described above (54, 41). Single chain K9/9 and K9/9 fusionproteins, but not sCrry, specifically targeted to rat kidneys and wasdetectable at 48 hr after administration (FIG. 28). Biodistribution at24 hr after administration was similar, and there was no radiolabelremaining in the blood at 24 hr.

In therapeutic studies, groups of 4 rats received PAN (150 mg/kg) at day0 and either PBS or complement inhibitor (40 mg/kg) on days 4,7 and 10.Urine (metabolic cages) and blood was collected and animals sacrificedon day 11. PAN treatment significantly impaired renal function asmeasured by creatinine clearance (FIG. 29, second bar from left). Therewas a slight, but not significant improvement in renal function in ratsreceiving sCrry therapy. However, creatinine clearance was significantlyimproved in PAN treated rats receiving either targeted Crry or CD59therapy (p<0.01). There was no significant difference in creatinineclearance between control (non-proteinuric) rats and PAN treated ratsreceiving either of the targeted inhibitors (FIG. 29). As expected,PAN-induced proteinuria was high in all rats whether treated withcomplement inhibitors or not (table 1). Kidney sections prepared fromrats treated with PAN and receiving no therapy showed dilation oftubular lumina and tubular and epithelial cell degeneration as assessedby loss of brush border (see FIG. 30 b, also in appendix). Minimalimprovement was seen with sCrry therapy (FIG. 30 d). In contrast tubulardilation and degeneration was significantly suppressed in PAN-treatedrats receiving targeted Crry and CD59 (FIG. 30 c shows K9/9-Crry, buthistology was indistinguishable with K9/9-CD59). These data demonstratetherapeutic efficacy of complement inhibition in this model, demonstratesignificant benefit of targeted vs untargeted complement inhibition anddirectly demonstrate an important role for MAC-mediated damage intubulointerstial injury induced by proteinuria. sCD59 is not aneffective inhibitor and this study demonstrates that appropriatelytargeted CD59 allows for the specific inhibition of the MAC in vivo.

In a separate experiment the circulatory half life of iodinatedrecombinant proteins was determined as described (54, 41, incorporatedherein by reference for the techniques taught therein). The half lives(t½) of the proteins were as follows: sCrry: 19 min, K9/9-Crry: 23 min,K9/9-CD59, 29 min, single chain K9/9: 21 min. To determine the effect ofthe recombinant proteins on systemic complement inhibition, rats wereinjected with proteins at 40 mg/Kg and blood collected at timescorresponding to 1, 3, 5 and 7×t½. Complement inhibitory activity inserum was determined, by measuring hemolytic activity (sensitized sheeperythrocytes). As expected, K9/9-CD59 had minimal inhibitory activity inserum (untargeted assay system) (FIG. 31). By about 3 hr (7×t½) afterthe injection of sCrry and K9/9-Crry, there was minimal complementinhibitory activity remaining in serum. The short t½ of targeted anduntargeted inhibitors, together with biodistribution data and the factthat sCrry is not protective, demonstrate that the kidney-boundcomplement inhibitors are effective at inhibiting complement locally andfor a prolonged period.

These data establish the use of targeted complement inhibitors in vivoand demonstrate important benefits of targeted versus untargetedsystemic complement inhibition in a model of disease. Although adifferent targeting vehicle is used in these studies (an antibodyfragment), the same principles apply for other targeting vehicles, suchas CR2.

4. Example 4

Disclosed herein are examples of constructs of the present inventionmade in accordance with the teaching herein. The terminology used hasthe following meaning: SCR=short consensus repeats; LP=Leader Peptide.The constructs all have the basic formula of CR2-linker-complementmodulator or complement modulator-linker-CR2. Notations in parenthesisindicate details within a particular section of the composition. Forexample, “(complete)” means that the entire mature protein is used inthe construct, whereas “(SCR2-4)” indicates that SCR1 is not part of theconstruct. It is understood that a linker can be a chemical linker, anatural liker peptide, or amino acid linking sequences (e.g.,(Gly₄Ser)₃). It is understood that this list is not limiting and onlyprovides examples of some of the constructs disclosed in the presentapplication.

CR2 (complete)-(Gly₄Ser)₃-DAF

CR2 (complete)-(Gly₄Ser)₃-human CD59

CR2 (complete)-(Gly₄Ser)₃-MCP

CR2 (complete)-(Gly₄Ser)₃-R1

CR2 (complete)-(Gly₄Ser)₃-Crry

CR2 (complete)-(Gly₄Ser)₃-mouse CD59

CR2 (complete)-(Gly₄Ser)₃-human IgG1 Fc

CR2 (complete)-(Gly₄Ser)₃-human IgM Fc

CR2 (complete)-(Gly₄Ser)₃-murine IgG3 Fc

CR2 (complete)-(Gly₄Ser)₃-murine IgM Fc

CR2 (complete)-(Gly₄Ser)₃-CVF

CR2 (complete)-(Gly₃Ser)₄-DAF

CR2 (complete)-(Gly₃Ser)₄-human CD59

CR2 (complete)-(Gly₃Ser)₄-MCP

CR2 (complete)-(Gly₃Ser)₄-CR1

CR2 (complete)-(Gly₃Ser)₄-Crry

CR2 (complete)-(Gly₃Ser)₄-mouse CD59

CR2 (complete)-(Gly₃Ser)₄-human IgG1 Fc

CR2 (complete)-(Gly₃Ser)₄-human IgM Fc

CR2 (complete)-(Gly₃Ser)₄-murine IgG3 Fc

CR2 (complete)-(Gly₃Ser)₄-murine IgM Fc

CR2 (complete)-(Gly₃Ser)₄-CVF

CR2 (complete)-(Gly₄Ser)₃-DAF (SCRs 2-4)

CR2 (complete)-(Gly₃Ser)₄-DAF (SCRs 2-4)

CR2 (complete)-(Gly₄Ser)₃-CR1 (LP-SCR1-4-SCR8-11-SCR15-18)

CR2 (complete)-(Gly₄Ser)₃-Crry (5 N-terminal SCRs)

CR2 (complete)-VSVFPLE-DAF

CR2 (complete)-VSVFPLE-human CD59

CR2 (complete)-VSVFPLE-MCP

CR2 (complete)-VSVFPLE-CR1

CR2 (complete)-VSVFPLE-Crry

CR2 (complete)-VSVFPLE-mouse CD59

CR2 (complete)-VSVFPLE-human IgG1 Fc

CR2 (complete)-VSVFPLE-human IgM Fc

CR2 (complete)-VSVFPLE-murine IgG3 Fc

CR2 (complete)-VSVFPLE-murine IgM Fc

CR2 (complete)-VSVFPLE-CVF

CR2 (complete)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-DAF

CR2 (complete)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-human CD59

CR2 (complete)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-MCP

CR2 (complete)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-CR1

CR2 (complete)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-Crry

CR2 (complete)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-mouse CD59

CR2 (complete)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-human IgG1Fc

CR2 (complete)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-human IgMFc

CR2 (complete)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-murine IgG3Fc

CR2 (complete)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-murine IgMFc

CR2 (complete)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-CVF

CR2 (complete)-bismaleimidohexane-DAF

CR2 (complete)-bismaleimidohexane-human CD59

CR2 (complete)-bismaleimidohexane-MCP

CR2 (complete)-bismaleimidohexane-CR1

CR2 (complete)-bismaleimidohexane-Crry

CR2 (complete)-bismaleimidohexane-mouse CD59

CR2 (complete)-bismaleimidohexane-human IgG1 Fc

CR2 (complete)-bismaleimidohexane-human IgM Fc

CR2 (complete)-bismaleimidohexane-murine IgG3 Fc

CR2 (complete)-bismaleimidohexane-murine IgM Fc

CR2 (complete)-bismaleimidohexane-CVF

CR2 (SCR1-2)-(Gly₄Ser)₃-DAF

CR2 (SCR1-2)-(Gly₄Ser)₃-human CD59

CR2 (SCR1-2)-(Gly₄Ser)₃-MCP

CR2 (SCR1-2)-(Gly₄Ser)₃-CR1

CR2 (SCR1-2)-(Gly₄Ser)₃-Crry

CR2 (SCR1-2)-(Gly₄Ser)₃-mouse CD59

CR2 (SCR1-2)-(Gly₄Ser)₃-human IgG1 Fc

CR2 (SCR1-2)-(Gly₄Ser)₃-human IgM Fc

CR2 (SCR1-2)-(Gly₄Ser)₃-murine IgG3 Fc

CR2 (SCR1-2)-(Gly₄Ser)₃-murine IgM Fc

CR2 (SCR1-2)-(Gly₄Ser)₃-CVF

CR2 (SCR1-2)-(Gly₃Ser)₄-DAF

CR2 (SCR1-2)-(Gly₃Ser)₄-human CD59

CR2 (SCR1-2)-(Gly₃Ser)₄-MCP

CR2 (SCR1-2)-(Gly₃Ser)₄-CR1

CR2 (SCR1-2)-(Gly₃Ser)₄-Crry

CR2 (SCR1-2)-(Gly₃Ser)₄-mouse CD59

CR2 (SCR1-2)-(Gly₃Ser)₄-human IgG1 Fc

CR2 (SCR1-2)-(Gly₃Ser)₄-human IgM Fc

CR2 (SCR1-2)-(Gly₃Ser)₄-murine IgG3 Fc

CR2 (SCR1-2)-(Gly₃Ser)₄-murine IgM Fc

CR2 (SCR1-2)-(Gly₃Ser)₄-CVF

CR2 (SCR1-2)-(Gly₄Ser)₃-DAF (SCRs 2-4)

CR2 (SCR1-2)-(Gly₃Ser)₄-DAF (SCRs 2-4)

CR2 (SCR1-2)-(Gly₄Ser)₃-CR1 (LP-SCR1-4-SCR8-11-SCR15-18)

CR2 (SCR1-2)-(Gly₄Ser)₃-Crry (5 N-terminal SCRs)

CR2 (SCR1-2)-VSVFPLE-DAF

CR2 (SCR1-2)-VSVFPLE-human CD59

CR2 (SCR1-2)-VSVFPLE-MCP

CR2 (SCR1-2)-VSVFPLE-CR1

CR2 (SCR1-2)-VSVFPLE-Crry

CR2 (SCR1-2)-VSVFPLE-mouse CD59

CR2 (SCR1-2)-VSVFPLE-human IgG1 Fc

CR2 (SCR1-2)-VSVFPLE-human IgM Fc

CR2 (SCR1-2)-VSVFPLE-murine IgG3 Fc

CR2 (SCR1-2)-VSVFPLE-murine IgM Fc

CR2 (SCR1-2)-VSVFPLE-CVF

CR2 (SCR1-2)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-DAF

CR2 (SCR1-2)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-human CD59

CR2 (SCR1-2)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-MCP

CR2 (SCR1-2)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-CR1

CR2 (SCR1-2)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-Crry

CR2 (SCR1-2)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-mouse CD59

CR2 (SCR1-2)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-human IgG1 Fc

CR2 (SCR1-2)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-human IgM Fc

CR2 (SCR1-2)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-murine IgG3Fc

CR2 (SCR1-2)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-murine IgM Fc

CR2 (SCR1-2)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-CVF

CR2 (SCR1-2)-bismaleimidohexane-DAF

CR2 (SCR1-2)-bismaleimidohexane-human CD59

CR2 (SCR1-2)-bismaleimidohexane-MCP

CR2 (SCR1-2)-bismaleimidohexane-CR1

CR2 (SCR1-2)-bismaleimidohexane-Crry

CR2 (SCR1-2)-bismaleimidohexane-mouse CD59

CR2 (SCR1-2)-bismaleimidohexane-human IgG1 Fc

CR2 (SCR1-2)-bismaleimidohexane-human IgM Fc

CR2 (SCR1-2)-bismaleimidohexane-murine IgG3 Fc

CR2 (SCR1-2)-bismaleimidohexane-murine IgM Fc

CR2 (SCR1-2)-bismaleimidohexane-CVF

CR2 (SCR1-3)-(Gly₄Ser)₃-DAF

CR2 (SCR1-3)-(Gly₄Ser)₃-human CD59

CR2 (SCR1-3)-(Gly₄Ser)₃-MCP

CR2 (SCR1-3)-(Gly₄Ser)₃-CR1

CR2 (SCR1-3)-(Gly₄Ser)₃-Crry

CR2 (SCR1-3)-(Gly₄Ser)₃-mouse CD59

CR2 (SCR1-3)-(Gly₄Ser)₃-human IgG1 Fc

CR2 (SCR1-3)-(Gly₄Ser)₃-human IgM Fc

CR2 (SCR1-3)-(Gly₄Ser)₃-murine IgG3 Fc

CR2 (SCR1-3)-(Gly₄Ser)₃-murine IgM Fc

CR2 (SCR1-3)-(Gly₄Ser)₃-CVF

CR2 (SCR1-3)-(Gly₃Ser)₄-DAF

CR2 (SCR1-3)-(Gly₃Ser)₄-human CD59

CR2 (SCR1-3)-(Gly₃Ser)₄-MCP

CR2 (SCR1-3)-(Gly₃Ser)₄-CR1

CR2 (SCR1-3)-(Gly₃Ser)₄-Crry

CR2 (SCR1-3)-(Gly₃Ser)₄-mouse CD59

CR2 (SCR1-3)-(Gly₃Ser)₄-human IgG1 Fc

CR2 (SCR1-3)-(Gly₃Ser)₄-human IgM Fc

CR2 (SCR1-3)-(Gly₃Ser)₄-murine IgG3 Fc

CR2 (SCR1-3)-(Gly₃Ser)₄-murine IgM Fc

CR2 (SCR1-3)-(Gly₃Ser)₄-CVF

CR2 (SCR1-3)-(Gly₄Ser)₃-DAF (SCRs 2-4)

CR2 (SCR1-3)-(Gly₃Ser)₄-DAF (SCRs 2-4)

CR2 (SCR1-3)-(Gly₄Ser)₃-CR1 (LP-SCR1-4-SCR8-11-SCR15-18)

CR2 (SCR1-3)-(Gly₄Ser)₃-Crry (5 N-terminal SCRs)

CR2 (SCR1-3)-VSVFPLE-DAF

CR2 (SCR1-3)-VSVFPLE-human CD59

CR2 (SCR1-3)-VSVFPLE-MCP

CR2 (SCR1-3)-VSVFPLE-CR1

CR2 (SCR1-3)-VSVFPLE-Crry

CR2 (SCR1-3)-VSVFPLE-mouse CD59

CR2 (SCR1-3)-VSVFPLE-human IgG1 Fc

CR2 (SCR1-3)-VSVFPLE-human IgM Fc

CR2 (SCR1-3)-VSVFPLE-murine IgG3 Fc

CR2 (SCR1-3)-VSVFPLE-murine IgM Fc

CR2 (SCR1-3)-VSVFPLE-CVF

CR2 (SCR1-3)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-DAF

CR2 (SCR1-3)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-human CD59

CR2 (SCR1-3)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-MCP

CR2 (SCR1-3)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-CR1

CR2 (SCR1-3)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-Crry

CR2 (SCR1-3)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-mouse CD59

CR2 (SCR1-3)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-human IgG1 Fc

CR2 (SCR1-3)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-human IgM Fc

CR2 (SCR1-3)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-murine IgG3Fc

CR2 (SCR1-3)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-murine IgM Fc

CR2 (SCR1-3)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-CVF

CR2 (SCR1-3)-bismaleimidohexane-DAF

CR2 (SCR1-3)-bismaleimidohexane-human CD59

CR2 (SCR1-3)-bismaleimidohexane-MCP

CR2 (SCR1-3)-bismaleimidohexane-CR1

CR2 (SCR1-3)-bismaleimidohexane-Crry

CR2 (SCR1-3)-bismaleimidohexane-mouse CD59

CR2 (SCR1-3)-bismaleimidohexane-human IgG1 Fc

CR2 (SCR1-3)-bismaleimidohexane-human IgM Fc

CR2 (SCR1-3)-bismaleimidohexane-murine IgG3 Fc

CR2 (SCR1-3)-bismaleimidohexane-murine IgM Fc

CR2 (SCR1-3)-bismaleimidohexane-CVF

CR2 (SCR1-4)-(Gly₄Ser)₃-DAF

CR2 (SCR1-4)-(Gly₄Ser)₃-human CD59

CR2 (SCR1-4)-(Gly₄Ser)₃-MCP

CR2 (SCR1-4)-(Gly₄Ser)₃-CR1

CR2 (SCR1-4)-(Gly₄Ser)₃-Crry

CR2 (SCR1-4)-(Gly₄Ser)₃-mouse CD59

CR2 (SCR1-4)-(Gly₄Ser)₃-human IgG1 Fc

CR2 (SCR1-4)-(Gly₄Ser)₃-human IgM Fc

CR2 (SCR1-4)-(Gly₄Ser)₃-murine IgG3 Fc

CR2 (SCR1-4)-(Gly₄Ser)₃-murine IgM Fc

CR2 (SCR1-4)-(Gly₄Ser)₃-CVF

CR2 (SCR1-4)-(Gly₃Ser)₄-DAF

CR2 (SCR1-4)-(Gly₃Ser)₄-human CD59

CR2 (SCR1-4)-(Gly₃Ser)₄-MCP

CR2 (SCR1-4)-(Gly₃Ser)₄-CR1

CR2 (SCR1-4)-(Gly₃Ser)₄-Crry

CR2 (SCR1-4)-(Gly₃Ser)₄-mouse CD59

CR2 (SCR1-4)-(Gly₃Ser)₄-human IgG1 Fc

CR2 (SCR1-4)-(Gly₃Ser)₄-human IgM Fc

CR2 (SCR1-4)-(Gly₃Ser)₄-murine IgG3 Fc

CR2 (SCR1-4)-(Gly₃Ser)₄-murine IgM Fc

CR2 (SCR1-4)-(Gly₃Ser)₄-CVF

CR2 (SCR1-4)-(Gly₄Ser)₃-DAF (SCRs 2-4)

CR2 (SCR1-4)-(Gly₃Ser)₄-DAF (SCRs 2-4)

CR2 (SCR1-4)-(Gly₄Ser)₃-CR1 (LP-SCR1-4-SCR8-11-SCR15-18)

CR2 (SCR1-4)-(Gly₄Ser)₃-Crry (5 N-terminal SCRs)

CR2 (SCR1-4)-VSVFPLE-DAF

CR2 (SCR1-4)-VSVFPLE-human CD59

CR2 (SCR1-4)-VSVFPLE-MCP

CR2 (SCR1-4)-VSVFPLE-CR1

CR2 (SCR1-4)-VSVFPLE-Crry

CR2 (SCR1-4)-VSVFPLE-mouse CD59

CR2 (SCR1-4)-VSVFPLE-human IgG1 Fc

CR2 (SCR1-4)-VSVFPLE-human IgM Fc

CR2 (SCR1-4)-VSVFPLE-murine IgG3 Fc

CR2 (SCR1-4)-VSVFPLE-murine IgM Fc

CR2 (SCR1-4)-VSVFPLE-CVF

CR2 (SCR1-4)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-DAF

CR2 (SCR1-4)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-human CD59

CR2 (SCR1-4)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-MCP

CR2 (SCR1-4)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-CR1

CR2 (SCR1-4)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-Crry

CR2 (SCR1-4)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-mouse CD59

CR2 (SCR1-4)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-human IgG1 Fc

CR2 (SCR1-4)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-human IgM Fc

CR2 (SCR1-4)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-murine IgG3Fc

CR2 (SCR1-4)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-murine IgM Fc

CR2 (SCR1-4)-m-Maleimidobenzoyl-N-hydroxysuccinimide ester-CVF

CR2 (SCR1-4)-bismaleimidohexane-DAF

CR2 (SCR1-4)-bismaleimidohexane-human CD59

CR2 (SCR1-4)-bismaleimidohexane-MCP

CR2 (SCR1-4)-bismaleimidohexane-CR1

CR2 (SCR1-4)-bismaleimidohexane-Crry

CR2 (SCR1-4)-bismaleimidohexane-mouse CD59

CR2 (SCR1-4)-bismaleimidohexane-human IgG1 Fc

CR2 (SCR1-4)-bismaleimidohexane-human IgM Fc

CR2 (SCR1-4)-bismaleimidohexane-murine IgG3 Fc

CR2 (SCR1-4)-bismaleimidohexane-murine IgM Fc

CR2 (SCR1-4)-bismaleimidohexane-CVF

G. REFERENCES

-   1. Wang, Y., Rollins, S. A., Madri, J. A., and Matis, L. A. 1995.    Anti-C5 monoclonal antibody therapy prevents collagen-induced    arthritis and ameliorates established disease. Proc Natl Acad Sci    USA 92:8955-8959.-   2. Wang, Y., Hu, Q., Madri, J. A., Rollins, S. A., Chodera, A., and    Matis, L. A. 1996. Amelioration of lupus-like autoimmune disease in    NZB/W F1 mice after treatment with a blocking monoclonal antibody    specific for complement component C5. Proc. Natl. Acad. Sci. USA    93:8563-8568.-   3. Kaplan, M. 2002. Eculizumab (Alexion). Curr Opin Investig Drugs    3:1017-1023.-   4. Whiss, P. A. 2002. Pexelizumab Alexion. Curr Opin Investig Drugs    3:870-877.-   5. Weisman, H. F., Bartow, T., Leppo, M. K., Marsh, H. C.,    Carson, G. R., Consino, M. F., Boyle, M. P., Roux, K. H.,    Weisfeldt, M. L., and Fearon, D. T. 1990. 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H. SEQUENCES

1. DAF

Nucleotide Sequence corresponds to SEQ ID NO: 1Amino Acid Sequence corresponds to SEQ ID NO: 2

2. CD59

Nucleotide Sequence corresponds to SEQ ID NO: 3Amino Acid Sequence corresponds to SEQ ID NO: 4

3. CR2-DAF

Nucleotide Sequence corresponds to SEQ ID NO: 5Amino Acid Sequence corresponds to SEQ ID NO: 6

4. CR2-Human CD59

Nucleotide Sequence corresponds to SEQ ID NO: 7Amino Acid Sequence corresponds to SEQ ID NO: 8

5. DAF-CR2

Nucleotide Sequence corresponds to SEQ ID NO: 9Amino Acid Sequence corresponds to SEQ ID NO: 10

6. Human CD59-CR2

Nucleotide Sequence corresponds to SEQ ID NO: 11Amino Acid Sequence corresponds to SEQ ID NO: 12

7. CR1

Nucleotide Sequence corresponds to SEQ ID NO: 13Amino Acid Sequence corresponds to SEQ ID NO: 14

8. MCP

Nucleotide Sequence corresponds to SEQ ID NO: 15Amino Acid Sequence corresponds to SEQ ID NO: 16

9. Mouse Crry

Amino Acid Sequence corresponds to SEQ ID NO: 17

10. Human IgG1 Fc

Amino Acid Sequence corresponds to SEQ ID NO: 18

11. Human IgM Fc

Amino Acid Sequence corresponds to SEQ ID NO: 19

12. CR2-Human IgG1 Fc

Nucleotide Sequence corresponds to SEQ ID NO: 20Amino Acid Sequence corresponds to SEQ ID NO: 21

13. Mouse IgG3 Fc

Amino Acid Sequence corresponds to SEQ ID NO: 22

14. Cobra Venom Factor

Nucleotide Sequence corresponds to SEQ ID NO: 23Amino Acid Sequence corresponds to SEQ ID NO: 24

15. Human CR2

Nucleotide Sequence corresponds to SEQ ID NO: 25Amino Acid Sequence corresponds to SEQ ID NO: 26

16. Mouse CR2

Nucleotide Sequence corresponds to SEQ ID NO: 27Amino Acid Sequence corresponds to SEQ ID NO: 28

17. Human CR2

Amino Acid Sequence corresponds to SEQ ID NO: 29

1. A composition comprising a construct, wherein the constructcomprises: (a) a CR2 or a fragment thereof, wherein the fragmentcontains at least the first two N-terminal SCR domains of the CR2protein; and (b) a modulator of complement activity.
 2. The compositionof claim 1, wherein the construct is a fusion protein.
 3. (canceled) 4.The composition of claim 1, wherein the modulator of complement activitycomprises a complement inhibitor.
 5. The composition of claim 4, whereinthe complement inhibitor comprises the first four SCR domains of decayaccelerating factor (DAF).
 6. The composition of claim 4, wherein thecomposition comprises SEQ ID NO.
 10. 7. The composition of claim 4,wherein the composition comprises SEQ ID NO.
 6. 8. The composition ofclaim 4, wherein the complement inhibitor comprises CD59.
 9. Thecomposition of claim 8, wherein the composition comprises SEQ ID NO. 12.10. The composition of claim 8, wherein the composition comprises SEQ IDNO.
 8. 11-14. (canceled)
 15. The composition of claim 4, wherein thecomplement inhibitor comprises Crry.
 16. The composition of claim 15,wherein the complement inhibitor comprises SEQ ID NO.
 17. 17. Thecomposition of claim 8, wherein the complement inhibitor is murine CD59or human CD59.
 18. (canceled)
 19. The composition of claim 1, whereinthe modulator of complement activity comprises a complement activator.20-26. (canceled)
 27. The composition of claim 19, wherein thecomplement activator comprises CVF.
 28. The composition of claim 27,wherein the complement activator comprises SEQ ID NO.
 24. 29. Thecomposition of claim 1, wherein the construct is an immunoconjugate. 30.A method of treating a condition affected by complement in a subjectcomprising administering to the subject the composition of any of claims1, 2, 4, 19, or
 52. 31. The method of claim 30, wherein the condition isa cancer.
 32. (canceled)
 33. The method of claim 30, wherein thecondition is selected from the group consisting of a viral infection, abacterial infection, a parasitic infection, and a fungal infection.34-40. (canceled)
 41. The method of claim 30, wherein the condition isan inflammatory condition. 42-45. (canceled)
 46. A method of reducingcomplement-mediated damage comprising administering to a subject thecomposition of any of claims 1, 2, 4, or
 52. 47. A method of enhancingcomplement-mediated damage comprising administering to a subject thecomposition of any of claims 1, 2, 19 or
 52. 48. The composition ofclaim 2, wherein the CR2 or a fragment thereof is fused to theN-terminus of the modulator of complement activity.
 49. The compositionof claim 2, wherein the CR2 or a fragment thereof is fused to theC-terminus of the modulator of complement activity.
 50. The compositionof claim 1, wherein the CR2 or a fragment thereof comprises afull-length CR2 protein.
 51. The composition of claim 1, wherein the CR2or a fragment thereof comprises the four N-terminal SCR domains of theCR2 protein.
 52. The composition of claim 1, wherein the modulator ofcomplement activity is selected from the group consisting of Crry, CD59,DAF, CVF, and a fragment thereof.
 53. The composition of claim 4,wherein the complement inhibitor comprises the first five N-terminal SCRdomains of Crry.
 54. The composition of claim 4, wherein the complementinhibitor comprises the extracellular region of CD59.
 55. A method oftargeting a modulator of complement activity to a site of complementactivation in a subject by administering to the subject a composition ofany of claims 1, 2, 4, 8, 17, 19 or
 52. 56. A method of treating aninflammatory condition in a subject by administering to the subject acomposition of any of claims 1, 2, 4, 8, 17 or
 52. 57. The method ofclaim 56, wherein the inflammatory condition is stroke.
 58. The methodof claim 56, wherein the inflammatory condition is ischemia reperfusioninjury.
 59. A nucleotide sequence encoding a fusion protein of claim 2.60-73. (canceled)
 74. The composition of claim 19, wherein thecomplement activator is an immunoglobulin.
 75. The composition of claim74, wherein the immunoglobulin is IgG1 Fc.
 76. The composition of claim74, wherein the immunoglobulin is IgM Fc.
 77. The composition of claim74, wherein the immunoglobulin is IgG3 Fc.
 78. The method of claim 30,comprising administering to the subject the composition of claim 19comprising a complement activator.
 79. The method of claim 78, whereinthe complement activator is an immunoglobulin.
 80. The method of claim79, wherein the immunoglobulin is an IgG1 Fc.
 81. The method of claim79, wherein the immunoglobulin is an IgM Fc.
 82. The method of claim 79,wherein the immunoglobulin is an IgG3 Fc.